WO2001090408A2 - Method for detecting and characterising activity of proteins involved in lesion and dna repair - Google Patents

Method for detecting and characterising activity of proteins involved in lesion and dna repair Download PDF

Info

Publication number
WO2001090408A2
WO2001090408A2 PCT/FR2001/001605 FR0101605W WO0190408A2 WO 2001090408 A2 WO2001090408 A2 WO 2001090408A2 FR 0101605 W FR0101605 W FR 0101605W WO 0190408 A2 WO0190408 A2 WO 0190408A2
Authority
WO
WIPO (PCT)
Prior art keywords
dna
repair
damaged
lesions
marker
Prior art date
Application number
PCT/FR2001/001605
Other languages
French (fr)
Other versions
WO2001090408A3 (en
Inventor
Sylvie Sauvaigo
Original Assignee
Commissariat A L'energie Atomique
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Commissariat A L'energie Atomique filed Critical Commissariat A L'energie Atomique
Priority to JP2001586602A priority Critical patent/JP4871481B2/en
Priority to DE60129220T priority patent/DE60129220T2/en
Priority to EP01938324A priority patent/EP1283911B1/en
Publication of WO2001090408A2 publication Critical patent/WO2001090408A2/en
Publication of WO2001090408A3 publication Critical patent/WO2001090408A3/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the subject of the present invention is a method for detecting and characterizing the activity of proteins involved in the repair of DNA damage. It applies in particular to the study of enzymes and protein factors involved in DNA repair, as well as to the study of the genotoxicity of chemicals or physical agents in living systems.
  • repair mechanisms Depending on the nature of the lesion, different repair mechanisms come into play and may even be in competition. Three main repair mechanisms can be distinguished: repair of mismatches, repair by nucleotide excision (REN) and repair by base excision (REB). On the other hand, homologous recombination which is a cellular mechanism intervening during meiosis, allows the repair of double strand breaks in DNA.
  • REN nucleotide excision
  • REB base excision
  • REN nucleotide excision
  • the base excision repair (REB) mechanism uses a family of enzymes called glycosylases. These are sometimes specific to the substrate to be excised, but in general recognize categories of modifications. Some glycosylases also have endonuclease activities. These enzymes repair oxidized, fragmented or allylated bases. This system also supports abasic sites, certain mismatches, certain single-strand breaks.
  • glycosylases In humans, six glycosylases have been identified (Wilson III, DM and Thompson, LH, 1997, Proc. Natl. Acad. Sci., 94, pages 12754-12757 [9]), which are mainly used to repair deaminated bases , oxidized, alkylated, or else to correct certain mismatches.
  • the action of the glycosylases results in the formation of an abasic site recognized by a specific endonuclease which incises the phosphodiester bond in 5 'of the eliminated base.
  • a poly erase replaces the eliminated nucleotide and a ligase closes the nucleotide chain.
  • glycosylases have an associated lyase activity which incises the phosphodiester bond at 3 'of the lesion and leads to the formation of a 5'-phosphate end.
  • REB pathway in which a small DNA fragment containing the damage is eliminated and then replaced. This could relate to some single strand breaks in DNA.
  • Specific enzymes encoded by identified genes are thus associated with the excision of defined lesions.
  • the procedures for evaluating cell repair activities are complicated and heavy to implement. They often require the use of chemically modified plasids.
  • the repair capacity of cell extracts is measured in vi tro by the repair of these modifications correlated with the incorporation of labeled nucleotides, during the resynthesis of the excised strands.
  • the method was initially developed by Wood et al., Cell, 53, 1988, pages 97-106 [1].
  • the supercoiled plasmids are modified by type C UV radiation, acetylaminofluorene or cis-dichlorodiamnineplatin.
  • the plasmids are then incubated in a medium containing cellular extracts, the four deoxynucleosidetriphosphates, one of which is marked in position: by a P, ATP and an ATP regeneration system. During incubation, lesions can be eliminated by the nucleotide or base excision systems.
  • the rate of DNA resynthesis is determined after migration of the DNA on agarose gel and counting of the radioactivity of the sought band.
  • Document FR-A-2 731 711 [3] describes a method for detecting DNA lesions which consists in fixing DNA on a solid phase, then in creating lesions on this DNA by means of an injurious product and to use cell extracts containing repair factors and a marker.
  • the purpose of this system is to allow the measurement of the genotoxic effects of certain chemical substances.
  • the DNA in the form of plasmids is first fixed on a solid phase (microplate well), then the plasmids are chemically modified.
  • the repair reactions are carried out in the presence of modified nucleoside triphosphates allowing non-radioactive detection of the neo-synthesized strands. This system does not allow specific modifications to be incorporated in the desired location.
  • the oligonucleotide containing the chemical modification (s) to be studied is labeled radioactive at one of its ends.
  • the action of the enzyme (kinetics of excision, determination of the affinity constants, cleavage or not of the oligonucleotide fragment, action on a single or double strand) is analyzed after electrophoresis on acrylamide gel.
  • Another technique avoids the use of radioactivity but requires a significant investment: it is the analysis of digestion by mass spectrometry (MALDI-TOF). This analysis is interesting because it allows the study of the excision mechanism. On the other hand, it is absolutely not suitable for routine analysis of an enzymatic activity.
  • the excision of the modified bases can also be followed by gas chromatography coupled with mass spectrometry. The demonstration was carried out using the excision of 5-hydroxy-5, 6-dihydrothymine and 5, 6-dihydrothymine by the endonuclease III on DNA irradiated by ⁇ radiation.
  • this precise technique has several drawbacks: it requires significant investment in analytical equipment, is not very sensitive, requires a lot of raw material and is not suitable for routine analyzes.
  • the subject of the invention is precisely a method of detecting and characterizing the activities of proteins involved in the repair of DNA damage, easier to implement, adaptable to the various repair modes, usable in routine easily. and fast, avoiding the use of radioactivity.
  • the method of detection and characterization of one or more protein activity (s) involved in the repair of 1 ⁇ DNA comprises the following steps: a) fix on a solid support at least one damaged DNA comprising at least one known lesion, b) subjecting this damaged DNA to the action of a repair composition containing or not at least one protein intervening for the repair of this damaged DNA, and c) determining the activity of this (these) protein (s) for repair by measuring the variation of the signal emitted by a marker which fixes on or is eliminated from the support during step b).
  • the damaged DNA of which at least one lesion is known to be fixed on a solid support such as a biochip then it is subjected to the action of a protein such as a repair enzyme, or '' a composition which could contain this protein, and the repair is followed by means of a marker which can be initially fixed on the DNA damaged or introduced into it during the repair process.
  • a protein such as a repair enzyme, or '' a composition which could contain this protein
  • the method can be used to characterize the proteins involved in DNA repair. It can also be used to demonstrate the non-functionality of certain proteins with respect to known lesions, or even to detect the absence of DNA repair proteins in compositions which normally should contain them.
  • repair genes are mutated and the enzymatic activity or the associated protein are not functional or are partially functional (xeroderma pigmentosum for example).
  • the solid support comprises at least one defined binding site for a damaged DNA fragment and advantageously several binding sites allowing the immobilization of different previously selected damaged DNA fragments.
  • the proteins whose activity we want to measure are the proteins involved in the repair process which generally involves recognition of the damage, an incision in the DNA chain, excision of the damage or fragments of nucleic acids, an in synthesis situ of DNA, and a ligation of the newly formed strand.
  • the proteins involved in this process can be chosen from: the proteins involved in the recognition of lesions such as XPA, the transcription factor TFIIH and the polypeptides which compose it, XPC, XPF, XPG and their associated proteins (ERCC family, etc.), HSSB, etc. (see Sancar, A. (1995) Annu. Rev. Genetics, 29, pages 69-105, [10]), proteins involved in the excision of lesions such as glycosylases, - proteins involved in the resynthesis of the nucleotide (s) excised strand such as polymerases, and proteins involved in the ligation of newly formed strands such as ligases.
  • the marker is present on the damaged DNA fixed on the support and it is eliminated by the action of the protein in step b).
  • This embodiment can be used for example to determine the activity of enzymes for incision or excision of damaged DNA lesions.
  • the marker may be present on one end of the damaged DNA.
  • an incision or an excision causes the elimination of the damaged DNA fragment carrying the marker and the loss of the signal on the support at the place where the damaged DNA is fixed.
  • the first embodiment can also be implemented with a specific marker for damaged DNA lesions such as an antibody, which reveals these lesions before step b). After action of the protein and repair of lesions, this marker cannot bind and a loss of the marker signal representative of the activity of the repair protein can be observed.
  • a specific marker for damaged DNA lesions such as an antibody
  • the ratio of the signal emitted by the specific antibodies before the DNA repair to the signal emitted by the antibodies after reaction of the protein, for example enzymatic reaction is correlated with the rate of DNA repair. In the latter case, compensation for damage is assessed by the disappearance of a specific damage signal.
  • the marker is present in the repair composition and is introduced into the DNA fixed on the support during step b).
  • modified nucleotides incorporated by the polymerases and which can be used for labeling the newly formed strand.
  • These modified nucleotides can carry a biotin, a hapten, a fluorescent compound or any other molecule compatible with the labeling of nucleic acids.
  • the markers that can be used to determine the activity of proteins involved in DNA repair can be of different types as long as they emit a detectable signal or can be revealed by emitting a detectable signal.
  • Several markers or mode of development can be used simultaneously or in a way sequential so as to highlight changes in state which have occurred in damaged DNA fragments, by the protein activities linked to repair.
  • the marker can in particular be an affinity molecule, a fluorescent compound, an antibody, a hapten or a biotin.
  • marker or marker revealer of fluorescent compounds with direct fluorescence or with indirect fluorescence.
  • Such molecules may for example be avidin, revealed by streptavidin-phycoerithrin, europium cryptates, fluorescent compounds such as fluoresceins, rhodamine, etc.
  • the energy transfer properties between fluorescent molecules can also be exploited to quantify an enzymatic activity involved in repair on a substrate oligonucleotide.
  • the oligonucleotide comprising the damaged base (s) also carries a fluorescent molecule authorizing an energy transfer with another fluorescent molecule located either on the same oligonucleotide or on an oligonucleotide in contact with the first, the emission of a signal after excitation proves the presence of the lesion on the support.
  • the incision of the oligonucleotide comprising the lesion (s) by a repairing enzyme leads to the elimination of the incised fragment and to the loss of the fluorescent signal, the energy transfer being unable to take place. This change in signal can be measured and correlated with a specific digestion rate.
  • the fixing of the damaged DNA to the solid support can be carried out by conventional methods.
  • damaged DNA can be synthesized directly on the solid support by means of automatic synthesizers allowing for example the incorporation of modified bases.
  • the damaged DNA can also be fixed on the solid support by any method compatible with maintaining the integrity of the DNA fragment containing the damage, for example by hybridization with another DNA fragment immobilized on the support, by immobilization by an affinity molecule, or by direct attachment to the chip by deposition or any other means.
  • a biochip is used as solid support on which are fixed, at determined locations, DNA fragments comprising lesions, identical or different, perfectly identified, or comprising multiple lesions which are not perfectly identified.
  • the DNA fragments comprising the lesions and immobilized can be short oligonucleotides
  • oligonucleotides comprising lesions in long DNA fragments:
  • the fragments immobilized DNA can be in the form of single or double strand.
  • the binding of the modified DNA can be done directly or via an intermediate molecule (biotin type, antigen, nucleic acid, etc.).
  • Long DNA fragments can also be damaged by any physical or chemical treatment that induces lesions. For example, they can be irradiated by radioactive, solar or ultraviolet radiation. They can be damaged by photosensitization reactions, by chemical agents, by carcinogens. In this case, the lesions will be induced statistically along the nucleotide chain. The quality, specificity and quantity of the lesions induced depends on the choice of the chemical or physical agent causing the lesions. We can thus target the category of lesions that we wish to introduce into a DNA fragment.
  • the support can for example be a biochip comprising DNA fragments, on which the damaged DNA is fixed by hybridization by means of an oligonucleotide comprising a complementary part of the damaged DNA and a complementary part of one of the fragments DNA from the biochip.
  • the invention benefits inter alia from the advantages of the chemical synthesis of oligonucleotides and therefore from the fact that one can choose exactly the sequences containing and surrounding the damage.
  • the same support can be functionalized by nucleotide fragments of identical or different sequences. Damage can be located at various locations, defined or not, of the sequences.
  • the DNA fragments comprising the lesions or else the complementary fragments of the fragments comprising the lesions can also contain markers useful for the detection of DNA transformations linked to an enzymatic repair activity. These markers can be affinity molecules, fluorescent compounds, or any specific detection system linked to a specific type of chip.
  • the reference chip or else a chip comprising damaged DNA fragments identical to those of the reference chip, known as the test chip, is incubated in the presence of solutions capable of containing the enzymatic repair activities.
  • the modified DNA fragments attached to the chip are susceptible to transformation by the enzymatic activities present.
  • a second reading of the test chip or of the reference chip is carried out under appropriate conditions and after the necessary disclosure steps.
  • the studs of the chip can thus emit signals different from the first reading, signifying that one or more enzymatic reactions have taken place on the chip.
  • the measurement of the transformation of DNA damaged by enzymes linked to repair systems can be followed by continuous recording of the signals emitted by the various studs of the chip, if the markers used are compatible with such a detection system. .
  • the method of the invention can be used for the study of enzymes or purified protein factors, as well as for the study of activities present in cellular extracts.
  • the miniaturization of the support improves the sensitivity of the system, in particular when using cell extracts.
  • the invention facilitates the detection of the various proteins linked to repair systems from cultured cells or from biopsies. Furthermore, by using a biochip comprising several damaged DNA sequences presenting different known lesions, it is possible to detect the repair capacities of these different damages for a limited quantity of the same cell extract. This is very important, especially in the case of diagnosis of certain genetic pathologies and diseases.
  • the invention has in particular many advantages.
  • the invention provides for the first time a global approach to the evaluation of the functionality of repair systems with access to several lesions and several repair mechanisms or stages simultaneously.
  • a direct analysis of the signals emitted by the various studs on the chip after the action of the enzymatic systems to be studied makes it possible to go back directly to the stages of the various repair systems and to conclude on their operation.
  • the invention allows the determination of a particular protein involved in the repair of a targeted lesion, such as the determination of a glycosylase involved in the excision of oxidized bases.
  • the lesion known to be the substrate preferential of the targeted glycosylase will be incorporated into a synthetic oligonucleotide and fixed on the chip at a determined location. It can therefore be seen that, by fixing several oligonucleotides comprising targeted and different lesions at different locations on the chip, we can have an evaluation of the functionality of the different glycosylases involved in the REB system.
  • the invention also allows the assay of proteins involved in steps common to the repair of all lesions such as polymerases and ligases. In the latter case, it is possible to use DNA fragments comprising lesions which are less precisely identified.
  • the invention is also characterized by the fact that the choice of the different substrates fixed in different pads of the chip makes it possible to target the repair system in which one is interested. Indeed, the fixation of short oligonucleotides (less than 25 bases) comprising lesions makes it possible to target the proteins of the REB system. The fixation of longer fragments (greater than 50 bases) comprising the lesions makes it possible to target both the proteins of the REB system and REN. The precise selection of fixed damages also makes it possible to target the repair systems to be studied.
  • the method of the invention can be used for the study of the specificity and the functionality of the proteins involved in repair with respect to known DNA lesions, to follow the kinetics of repair of lesions of the DNA by proteins, to assess the genotoxic effect of substances or physical agents inhibiting or stimulating the synthesis of proteins' involved in DNA repair, or for the diagnosis of deficiencies in repair proteins linked to diseases.
  • Other characteristics and advantages of the invention will appear better on reading the following examples given, of course, by way of illustration and not limitation with reference to the appended drawings.
  • FIG. 1 schematically represents the state of a system in the first step of the method of the invention.
  • FIG. 2 schematically represents the state of the system of FIG. 1 after step b) of the method of the invention.
  • the first embodiment of the method of the invention is used to determine a glycosylase-type repair activity on a damaged oligonucleotide comprising as lesion an 8-oxo-7,8 dihydro-2 '-deoxyguanosine.
  • MICAM pb4 biochip obtained as described by Livache et al, Biosens.
  • Sequence H 5 'TTTTT CCA CAC GGT AGG TAT CAG TC
  • the functionalized part of the biochip is incubated in the presence of a hybrid formed from one damaged oligonucleotide (O), comprising an 8-oxo-7, 8-dihydro-2 '- deoxyguanosine, and an oligonucleotide (cOcH) comprising a complementary part of 1 Oligonucleotide O (cO) and a complementary part of oligonucleotide H fixed on the chip (cH).
  • the oligonucleotide O has a biotin at its 3 'end.
  • cOcH 5 'GCA GCC CGG GGG ATC CAC TAG TTC GAC TGA CTA CCG TGT GG
  • the O-cOcH hybrid is obtained by incubation for 60 min at 37 ° C of 10 pmol of cOcH and 12 pmol of O in 200 ⁇ l of PBS buffer containing 0.2 M NaCl. 20 ⁇ l of this solution are removed and added to the functionalized part of the chip which forms a cuvette. After 1 hour incubation in a humid environment at 37 ° C., the chip is soaked in a washing buffer (PBS / 0.2 M NaCl / 0.05% tween 20).
  • FIG. 1 represents the system obtained by fixing the oligonucleotide O on the pad 1 of the biochip comprising the oligonucleotide H.
  • This oligonucleotide H is hybridized with the sequence cH of The oligonucleotide 3 which also comprises a sequence hybridized with the damaged oligonucleotide O carrying a biotin 5 at its 3 'end and a lesion 7.
  • the oligonucleotide O containing the lesion and fixed on the biochip by hybridization is revealed after incubation for 10 min at room temperature with 20 ⁇ l of PBS / NaCl buffer containing 0.5% of bovine serum albumin BSA and 1 ⁇ l of streptavidin-phycoerythrin R (0.5 mg / ml; Jackson ImmunoResearch). After washing in PBS / NaCl / t een, the biochip is observed under a fluorescence microscope and the signal is analyzed with Image Pro Plus software.
  • the signal is integrated in pixels.
  • the functionalized studs emit a slight fluorescent signal even in the absence of any hybridization.
  • the non-functionalized studs are black.
  • Hybridization of one oligonucleotide with DNA damage is therefore specific.
  • the biochip is washed with 0.1 N NaOH for 10 min, then rinsed for 10 min with H 2 0, which has the effect of removing oligonucleotides 0 and cOcH from the biochip.
  • H 2 0.1 N NaOH
  • the chip is again hybridized with the O-cOcH hybrid under the conditions defined above. After washing with PBS / NaCl / tween, the chip is balanced for 10 min with a 0.1 M RCl buffer, Tris, then 20 ⁇ l of this same buffer containing 0.5 ⁇ g of Fapy DNA Glycosylase (S. Boiteux, CEA Fontenay- aux-Roses, France) are added. The chip is incubated for 30 min at 37 ° C. in a humid atmosphere. After washing with PBS / NaCl / tween, the revelation step is carried out as above with the streptavidin-phycoerythrin R. The signal is recorded after washing with PBS / tween. The intense fluorescence on the H stud has disappeared. The signal to noise ratio (H / I) is 1.04.
  • FIG. 2 represents the state of the system after reaction with the glycosylase type enzyme.
  • the oligonucleotide 0 has been eliminated from the biochip.
  • the enzyme Fapy DNA glycosylase cut the oligonucleotide O at the level of 8-oxo-7, 8-dihydro-deoxyguanosine.
  • the short DNA fragments thus generated form hybrids which are unstable under the conditions used and are eliminated from the biochip. This results in the disappearance of the fluorescent signal due to the revelation of biotin.
  • This example illustrates the detection of a glycosylase type activity in a cell lysate using the first embodiment of the method of the invention.
  • the oligonucleotide O used in example 1, comprising the 8-oxo-7,8-dihydro-2 '- deoxynucleotide, is hybridized as in example 1 on the biochip pb4, which corresponds to the system shown in figure 1.
  • a total cell lysate is prepared from Hela cells in culture.
  • the cells are trypsinized and then washed in PBS buffer.
  • the pellet containing approximately 15.10 cells is taken up in 1 ml of lysis buffer
  • Phenylmethylsulphonyl Fluoride (PMSF, Sigma, 17.4 mg / l in isopropanol). The cells are ground, then the lysate is centrifuged at 4 ° C, for 50 min at
  • the supernatant is recovered and then 200 ⁇ l of glycerol and 20 ⁇ l of 0.1M dithiotreitol are added.
  • the lysates are aliquoted and stored at -80 ° C.
  • the biochip is then incubated with 20 ⁇ l of cell extract for 45 min at 37 ° C.
  • the revelation is carried out as in Example 1.
  • the signal from pad H is recorded. It is 180 pixels.
  • the experiment is repeated with lysate denatured (heating at 100 ° C for 10 min).
  • the signal from pad H is saturated at 250 pixels. There is therefore a loss of signal following the incubation of the biochip functionalized by the duplex containing the damage with cell lysate br t. This loss of signal is approximately 30%, which corresponds to the partial cleavage of the oligonucleotide modified 0 by the enzymes contained in the lysate.
  • This example illustrates the detection of the excision / resynthesis repair activity of a total cell lysate, using a modified DNA fragment fixed on a microcarrier.
  • a DNA fragment of 5000 base pairs is prepared by PCR amplification from phage lambda using the "Expand TM Long template PCR System” kit from Roche.
  • One of the amplification primers has a 15 base sequence (J 15 ) at its 5 'end separated by an amino synthon from the sequence hybridizing on the phage. This sequence remains single stranded after PCR amplification.
  • This PCR fragment is purified by an S300 micro-spin column (Amersham-Pharmacia). The DNA strand is irradiated for 3 minutes with ultraviolet rays of type C ( ⁇ ax 254 nm, 0.8 J / cm 2 ).
  • the irradiated DNA is then hybridized by via an oligonucleotide of 30 bases long cIi 5 cJ 15 complementary for part of the sequence I 15 and for another part of the sequence J 15 , on a pb2 biochip (MICAM) comprising four different oligonucleotide sequences out of four different studs (SI, S2, S3, I 15 ), including the sequence called I 15 .
  • I 15 5 'TTTTT ATC CGT TCT ACA GCC
  • the hybridization takes place for 1 hour at 37 ° C. in 20 ⁇ l of PBS / NaCl buffer containing approximately 0.1 pmole of the PCR amplification product.
  • the chip is then incubated in the presence of total cell extracts in a suitable buffer as described below.
  • a solution is prepared containing 25 ⁇ l of Hela cell cell lysate, 10 ⁇ l of 5X reaction buffer (Hepes KOH 225 mM pH 7.8, KCl, 350 mM, MgCl 2 37.5 mM, DTT 4.5 mM, EDTA 2 mM, BSA 0.09 mg / ml, glycerol 17%), ATP 2 mM, dGTP 5 ⁇ , dATP 5 ⁇ M, dCTP 5 ⁇ M, dTTP 1 ⁇ M, Biotin-16-2 '- deoxyuridine-5' - triphosphate 4 ⁇ M , 200 mM phosphocreatine, creatine phosphokinase type I 12.5 ⁇ g in a total volume of 50 ⁇ l.
  • 5X reaction buffer Hepes KOH 225 mM pH 7.8, KCl, 350 mM, MgCl 2 37.5 mM, DTT 4.5 mM, EDTA 2 mM
  • the chip is then analyzed under a microscope.
  • a strong fluorescence (saturated signal) is observed at the level of the pad functionalized by the oligonucleotide I 15
  • the signal to noise ratio (I 15 / S3) is 2.2.
  • a streptavidin-phycoerythin revelation carried out before repair, by the enzymes contained in the lysate, of the modified DNA fragment gives a signal to noise ratio (I 15 / S3) of 1.
  • This example shows that the enzymatic activities involved in repairing the modified bases of DNA are detectable on a chip functionalized by a DNA fragment comprising modified bases.
  • This example illustrates the use of specific antibodies for the demonstration of lesions constituted by pyrimidine dimers of cyclobutane type.
  • Example 3 The same biochip is used as in Example 3, to which the irradiated DNA is fixed under the same conditions as those of Example 3, using the same oligonucleotide cl 15 cJ 15 . After washing with PBS / NaCl / tween, the cyclobutane type pyrimidine dimers formed by UVB irradiation are revealed.
  • the chip is incubated with 20 ⁇ l of PBS / NaCl buffer containing 1 ⁇ l of anti-dimer antibody (500 ⁇ g / ml; Ka iya Biomedicam Company, Seattle, USA), for 1 hour at 37 ° C.
  • the chip After washing with PBS / NaCl / tween the chip is incubated in the presence of 20 ⁇ l of PBS / NaCl containing 1 ⁇ l of goat anti-mouse antibody coupled to the marker
  • the signal to noise ratio (I 15 / S3) is 1.33.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention concerns a method for detecting and characterising the activity of proteins(s) involved in DNA repair, comprising the following steps: a) fixing on a solid support (1) a known damaged DNA O comprising a lesion (7); b) subjecting said damaged DNA to the action of a composition containing at least a protein intervening in the repair of said damaged DNA; and c) determining the activity of said protein for repair by measuring the variation in the signal emitted by a marker (5) which fixes itself on or is eliminated from the support during step b).

Description

DETECTION ET CARACTERISATION DE L'ACTIVITE DETECTION AND CHARACTERIZATION OF ACTIVITY
DE PROTEINES IMPLIQUEES DANS LA REPARATIONPROTEINS INVOLVED IN REPAIR
DE LESIONS DE L'ADNOF DNA DAMAGE
DESCRIPTIONDESCRIPTION
Domaine techniqueTechnical area
La présente invention a pour objet un procédé de détection et de caractérisation de l'activité de protéines impliquées dans la réparation de lésions de l'ADN. Elle s'applique en particulier à l'étude des enzymes et des facteurs protéiques impliqués dans la réparation de l'ADN, ainsi qu'à l'étude de la génotoxicité de substances chimiques ou d'agents physiques dans des systèmes vivants.The subject of the present invention is a method for detecting and characterizing the activity of proteins involved in the repair of DNA damage. It applies in particular to the study of enzymes and protein factors involved in DNA repair, as well as to the study of the genotoxicity of chemicals or physical agents in living systems.
État de la technique antérieureState of the art
Des lésions ou dommages peuvent se former dans l'ADN cellulaire par exposition à divers agents chimiques ou physiques, en particulier après irradiation (rayonnements ionisants et solaires) , après induction par un stress oxydant, ou après exposition à certains agents génotoxiques ou cytotoxiques . Des nucleosides endommagés sont également susceptibles de s'accumuler naturellement dans le génome, au cours du processus de vieillissement cellulaire. Les lésions ou dommages des acides nucléiques sont de plusieurs types et sont souvent caractéristiques de l'agent qui les a induits. Ces dommages incluent des cassures simple et double-brin, des sites abasiques et des modifications de bases nucléiques .Lesions or damage can form in cellular DNA by exposure to various chemical or physical agents, in particular after irradiation (ionizing and solar radiation), after induction by oxidative stress, or after exposure to certain genotoxic or cytotoxic agents. Damaged nucleosides are also likely to accumulate naturally in the genome during the process of cell aging. Nucleic acid damage or damage is of many types and is often characteristic of the agent that caused it. These damage includes single and double-strand breaks, abasic sites and nucleic base modifications.
Suivant la nature de la lésion, différents mécanismes de réparation entrent en jeu et peuvent même être en compétition. On peut distinguer trois mécanismes principaux de réparation : la réparation des mésappariements, la réparation par excision de nucleotides (REN) et la réparation par excision de bases (REB). D'autre part, la recombinaison homologue qui est un mécanisme cellulaire intervenant lors de la méiose, permet la réparation des cassures double brins d'ADN.Depending on the nature of the lesion, different repair mechanisms come into play and may even be in competition. Three main repair mechanisms can be distinguished: repair of mismatches, repair by nucleotide excision (REN) and repair by base excision (REB). On the other hand, homologous recombination which is a cellular mechanism intervening during meiosis, allows the repair of double strand breaks in DNA.
Ces mécanismes font intervenir différentes protéines de réparation. Ainsi, le mécanisme de réparation par excision de nucleotides (REN) implique plusieurs étapes qui sont :These mechanisms involve different repair proteins. Thus, the repair mechanism by nucleotide excision (REN) involves several steps which are:
1) la reconnaissance du dommage par un complexe protéique, 2) l'incision du brin contenant la lésion de chaque coté de celle-ci,1) recognition of the damage by a protein complex, 2) the incision of the strand containing the lesion on each side thereof,
3) l'excision d'un fragment oligonucléotidique contenant la lésion, et3) excision of an oligonucleotide fragment containing the lesion, and
4) la synthèse du nouveau brin d'ADN intègre avec une ligation finale.4) the synthesis of the new DNA strand integrates with a final ligation.
De nombreuses protéines et enzymes sont impliquées dans ce mécanisme qui répare principalement les lésions photo-induites et les dommages volumineux provoqués par des traitements chimiques. Le mécanisme de réparation par excision de bases (REB) fait appel à une famille d'enzymes appelées glycosylases . Celles-ci sont parfois spécifiques du substrat à exciser, mais en général reconnaissent des catégories de modifications. Certaines glycosylases possèdent également des activités endonucléases . Ces enzymes réparent les bases oxydées, fragmentées ou al ylées . Ce système prend en charge, en outre, les sites abasiques, certains mésappariements, certaines cassures simple brin.Many proteins and enzymes are involved in this mechanism which mainly repairs photo-induced lesions and bulky damage caused by chemical treatments. The base excision repair (REB) mechanism uses a family of enzymes called glycosylases. These are sometimes specific to the substrate to be excised, but in general recognize categories of modifications. Some glycosylases also have endonuclease activities. These enzymes repair oxidized, fragmented or allylated bases. This system also supports abasic sites, certain mismatches, certain single-strand breaks.
Chez les humains, six glycosylases ont été identifiées (Wilson III, D.M. and Thompson, L.H., 1997, Proc. Natl. Acad. Sci . , 94, pages 12754-12757 [9]), qui servent principalement à la réparation de bases désaminées, oxydées, alkylées, ou bien à la correction de certains mésappariements. L'action des glycosylases résulte en la formation d'un site abasique reconnu par une endonuclease spécifique qui incise la liaison phosphodiester en 5' de la base éliminée. Une poly érase remplace le nucléotide éliminé et une ligase referme la chaîne nucléotidique . Il est à noter que certaines glycosylases possèdent une activité lyase associée qui incise la liaison phosphodiester en 3 ' de la lésion et conduit à la formation d'une extrémité 5'- phosphate. Il existe une alternative à cette voie de REB au cours de laquelle un petit fragment d'ADN contenant le dommage est éliminé, puis remplacé. Cela pourrait concerner certaines cassures simple brin de l'ADN. Des enzymes spécifiques codées par des gènes identifiés sont ainsi associées à l'excision des lésions définies. Les procédés destinés à évaluer les activités de réparation cellulaire sont compliqués et lourds à mettre en œuvre. Ils nécessitent souvent l'utilisation de plas ides modifiés chimiquement. La capacité de réparation d'extraits cellulaires est mesurée in vi tro par la réparation de ces modifications corrélée à l'incorporation de nucleotides marqués, lors de la resynthèse des brins excisés. La méthode a été initialement développée par Wood et al., Cell, 53, 1988, pages 97-106 [1]. Les plasmides superenroulés sont modifiés par des rayonnements UV de type C, 1 ' acétyl-aminofluorène ou bien le cis- dichlorodiamnineplatine . Les plasmides sont ensuite incubés dans un milieu contenant des extraits cellulaires, les quatre désoxynucléosidetriphosphates , dont un est marqué en position : par un P, de l'ATP et un système de régénération d'ATP. Pendant l'incubation, des lésions peuvent être éliminées par les systèmes d'excision de nucleotides ou d'excision de bases. Le taux de resynthèse d'ADN est déterminé après migration de l'ADN sur gel d'agarose et comptage de la radioactivité de la bande recherchée. L'importance et la spécificité de la réparation sont liées à la qualité des extraits et du plasmide, aux lésions créées et aux paramètres de réaction, comme il est décrit par Salles et al, Biochimie, 77, 1995, pages 796-802 [ 2 ] . En particulier, une préparation plasmidique initiale est nécessaire pour éliminer les plasmides contenant des cassures avant traitement ou des fragments de chromosome bactérien qui fourniraient -des .sites d'initiation de polymérisation. Un autre inconvénient de cette technique a trait aux lésions créées . Les traitements utilisés n'induisent pas un défaut unique. Par exemple, les dommages majoritaires engendrés par les rayonnements UVC sont les dimères de pyrimidine de type cyclobutane. Mais d'autres lésions sont formées comme les photoproduits, des cassures de chaînes, des hydrates de cytosine etc. Ainsi, un suivi particulier de la réparation de chacun des dommages ne peut pas être effectué facilement à 1 ' aide de ces substrats .In humans, six glycosylases have been identified (Wilson III, DM and Thompson, LH, 1997, Proc. Natl. Acad. Sci., 94, pages 12754-12757 [9]), which are mainly used to repair deaminated bases , oxidized, alkylated, or else to correct certain mismatches. The action of the glycosylases results in the formation of an abasic site recognized by a specific endonuclease which incises the phosphodiester bond in 5 'of the eliminated base. A poly erase replaces the eliminated nucleotide and a ligase closes the nucleotide chain. It should be noted that certain glycosylases have an associated lyase activity which incises the phosphodiester bond at 3 'of the lesion and leads to the formation of a 5'-phosphate end. There is an alternative to this REB pathway in which a small DNA fragment containing the damage is eliminated and then replaced. This could relate to some single strand breaks in DNA. Specific enzymes encoded by identified genes are thus associated with the excision of defined lesions. The procedures for evaluating cell repair activities are complicated and heavy to implement. They often require the use of chemically modified plasids. The repair capacity of cell extracts is measured in vi tro by the repair of these modifications correlated with the incorporation of labeled nucleotides, during the resynthesis of the excised strands. The method was initially developed by Wood et al., Cell, 53, 1988, pages 97-106 [1]. The supercoiled plasmids are modified by type C UV radiation, acetylaminofluorene or cis-dichlorodiamnineplatin. The plasmids are then incubated in a medium containing cellular extracts, the four deoxynucleosidetriphosphates, one of which is marked in position: by a P, ATP and an ATP regeneration system. During incubation, lesions can be eliminated by the nucleotide or base excision systems. The rate of DNA resynthesis is determined after migration of the DNA on agarose gel and counting of the radioactivity of the sought band. The importance and specificity of the repair are linked to the quality of the extracts and of the plasmid, to the lesions created and to the reaction parameters, as described by Salles et al, Biochemistry, 77, 1995, pages 796-802 [2 ]. In particular, an initial plasmid preparation is necessary to remove plasmids containing breaks before treatment or bacterial chromosome fragments which would provide polymerization initiation sites. Another disadvantage of this technique relates to the lesions created. The treatments used do not induce a single defect. For example, the major damage caused by UVC radiation is the cyclobutane type pyrimidine dimers. But other lesions are formed such as photoproducts, chain breaks, cytosine hydrates etc. Thus, particular monitoring of the repair of each of the damages cannot be easily carried out using these substrates.
Le document FR-A-2 731 711 [3] décrit un procédé de détection de lésions de l'ADN consistant à fixer l'ADN sur une phase solide, puis à créer des lésions sur cet ADN au moyen d'un produit lésant et à utiliser des extraits cellulaires contenant des facteurs de réparation et un marqueur. Le but de ce système est de permettre la mesure des effets génotoxiques de certaines substances chimiques. L'ADN sous forme de plasmides est tout d'abord fixé sur une phase solide (puits de microplaque) , puis les plasmides sont modifiés chimiquement. Les réactions de réparation sont effectuées en présence de nucleosides triphosphates modifiés permettant une détection non radioactive des brins néo-synthétisés . Ce système ne permet pas d'incorporer de façon ciblée, à l'endroit désiré des modifications spécifiques. Il s'agit d'un système qui permet la détection d'un effet global d'un agent modificateur de l'ADN sans identifier les lésions reconnues par les systèmes de réparation. Par ailleurs, ce procédé n'a pas pour but de détecter et quantifier l'activité des protéines impliquées dans la réparation de l'ADN, mais d'identifier la présence de lésions sur l'ADN traité. Page et al, Biochemistry, 29, 1990, pages 1016-1024 [4] et Huang et al, Proc . Natl . Acad. Sci., 91, 1994, pages 12213-12217, [5], utilisent un test différent afin de mesurer les capacités d'excision de dommages par des extraits cellulaires de diverses origines. Des lésions spécifiques sont créées ou incorporées dans de courts oligonucléotides de synthèse. Ceux-ci sont ensuite liés entre eux par des ligases pour donner des fragments doubles-brins plus longs (150 à 180 paires de bases). L'incorporation de 3 P en différentes positions des fragments permet de localiser les sites de coupure créés par les enzymes de réparation. L'analyse s'effectue par autoradiographie après electrophorese sur gel d ' acrylamide . Ce test est applicable aux réparations de type REB et REN.Document FR-A-2 731 711 [3] describes a method for detecting DNA lesions which consists in fixing DNA on a solid phase, then in creating lesions on this DNA by means of an injurious product and to use cell extracts containing repair factors and a marker. The purpose of this system is to allow the measurement of the genotoxic effects of certain chemical substances. The DNA in the form of plasmids is first fixed on a solid phase (microplate well), then the plasmids are chemically modified. The repair reactions are carried out in the presence of modified nucleoside triphosphates allowing non-radioactive detection of the neo-synthesized strands. This system does not allow specific modifications to be incorporated in the desired location. It is a system that allows the detection of an overall effect of a DNA modifying agent without identifying the lesions recognized by the repair systems. Furthermore, this process is not intended to detect and quantify the activity of proteins involved in DNA repair, but to identify the presence of lesions on the treated DNA. Page et al, Biochemistry, 29, 1990, pages 1016-1024 [4] and Huang et al, Proc. Natl. Acad. Sci., 91, 1994, pages 12213-12217, [5], use a different test in order to measure the capacities for excision of damage by cell extracts of various origins. Specific lesions are created or incorporated into short synthetic oligonucleotides. These are then linked together by ligases to give longer double-stranded fragments (150 to 180 base pairs). The incorporation of 3 P at different positions of the fragments makes it possible to locate the cleavage sites created by the repair enzymes. The analysis is carried out by autoradiography after electrophoresis on acrylamide gel. This test is applicable to REB and REN type repairs.
Tous les procédés décrits ci-dessus présentent des inconvénients. Dans la plupart des cas, des marquages radioactifs et des séparations longues par electrophorese sont nécessaires afin de déterminer l'excision des dommages. L'utilisation de plasmides est lourde à mettre en œuvre car des précautions sont nécessaires afin de s'assurer de leur pureté et minimiser le bruit de fond. D'autre part, comme on l'a mentionné, l'induction d'un seul type de dommage par un stress est impossible et plusieurs lésions sont ainsi présentes simultanément dans l'ADN.All the methods described above have drawbacks. In most cases, radioactive markings and long separations by electrophoresis are necessary to determine the excision of the damage. The use of plasmids is cumbersome to implement because precautions are necessary in order to ensure their purity and minimize the background noise. On the other hand, as mentioned, the induction of a single type of damage by stress is impossible and several lesions are thus present simultaneously in DNA.
On connaît aussi des procédés permettant d'étudier les spécificités et les mécanismes d'excision de dommages par des enzymes de réparation sur des oligonucléotides de synthèse de 15 à 50 bases de long contenant des lésions bien définies, comme il est décrit par Romieu et al, J. Org. Che . , 63, 1998, pages 5245-5249 [6] et par D'Ham et al, Biochemistry, 38, 1999, pages 3335-3344 [7].Also known are methods for studying the specificities and mechanisms of excision of damage by repair enzymes on synthetic oligonucleotides 15 to 50 bases long containing well defined lesions, as is described by Romieu et al, J. Org. Che. , 63, 1998, pages 5245-5249 [6] and by D'Ham et al, Biochemistry, 38, 1999, pages 3335-3344 [7].
Dans la plupart des cas, 1 'oligonucléotide contenant la ou les modifications chimiques à étudier est marqué radioactive ent à une de ses extrémités. L'action de l'enzyme (cinétique d'excision, détermination des constantes d'affinité, coupure ou non du fragment oligonucleotidique, action sur un simple ou un double brin) est analysée après electrophorese sur gel d' acrylamide.In most cases, the oligonucleotide containing the chemical modification (s) to be studied is labeled radioactive at one of its ends. The action of the enzyme (kinetics of excision, determination of the affinity constants, cleavage or not of the oligonucleotide fragment, action on a single or double strand) is analyzed after electrophoresis on acrylamide gel.
Une autre technique évite 1 ' emploi de la radioactivité mais nécessite un investissement important : c'est l'analyse de la digestion par spectrométrie de masse (MALDI-TOF) . Cette analyse est intéressante car elle permet l'étude du mécanisme d'excision. En revanche, elle n'est absolument pas adaptée à une analyse en routine d'une activité enzymatique . L'excision des bases modifiées peut être aussi suivie par chromatographie gazeuse couplée à la spectrométrie de masse. La démonstration a été réalisée à partir de l'excision de la 5-hydroxy-5, 6- dihydrothymine et de la 5 , 6-dihydrothymine par 1 ' endonuclease III sur de l'ADN irradié par rayonnement γ. Cette technique précise présente toutefois plusieurs inconvénients : elle demande un investissement en matériel analytique important, est peu sensible, nécessite beaucoup de matière première et n'est pas adaptée à des analyses de routine. Exposé de l'invention.Another technique avoids the use of radioactivity but requires a significant investment: it is the analysis of digestion by mass spectrometry (MALDI-TOF). This analysis is interesting because it allows the study of the excision mechanism. On the other hand, it is absolutely not suitable for routine analysis of an enzymatic activity. The excision of the modified bases can also be followed by gas chromatography coupled with mass spectrometry. The demonstration was carried out using the excision of 5-hydroxy-5, 6-dihydrothymine and 5, 6-dihydrothymine by the endonuclease III on DNA irradiated by γ radiation. However, this precise technique has several drawbacks: it requires significant investment in analytical equipment, is not very sensitive, requires a lot of raw material and is not suitable for routine analyzes. Statement of the invention.
L'invention a précisément pour objet un procédé de détection et de caractérisation d'activités de protéines impliquées dans la réparation de dommages de l'ADN, plus facile à mettre en œuvre, adaptable aux différents modes de réparation, utilisable en routine de manière aisée et rapide, en évitant l'emploi de la radioactivité .The subject of the invention is precisely a method of detecting and characterizing the activities of proteins involved in the repair of DNA damage, easier to implement, adaptable to the various repair modes, usable in routine easily. and fast, avoiding the use of radioactivity.
Selon l'invention, le procédé de détection et de caractérisation d'une ou plusieurs activité (s) de protéine (s) impliquée(s) dans la réparation de 1~ADN, comprend les étapes suivantes : a) fixer sur un support solide au moins un ADN endommagé comportant au moins une lésion connue, b) soumettre cet ADN endommagé à l'action d'une composition de réparation contenant ou non au moins une protéine intervenant pour la réparation de cet ADN endommagé, et c) déterminer l'activité de cette (ces) protéine (s) pour la réparation en mesurant la variation du signal émis par un marqueur qui se fixe sur ou s'élimine du support lors de l'étape b) .According to the invention, the method of detection and characterization of one or more protein activity (s) involved in the repair of 1 ~ DNA, comprises the following steps: a) fix on a solid support at least one damaged DNA comprising at least one known lesion, b) subjecting this damaged DNA to the action of a repair composition containing or not at least one protein intervening for the repair of this damaged DNA, and c) determining the activity of this (these) protein (s) for repair by measuring the variation of the signal emitted by a marker which fixes on or is eliminated from the support during step b).
Dans ce procédé, on fixe sur un support solide tel qu'une biopuce, l'ADN endommagé dont on connaît au moins une lésion, puis on le soumet à l'action d'une protéine telle qu'une enzyme de réparation, ou d'une composition qui pourrait contenir cette protéine, et on suit la réparation au moyen d'un marqueur qui peut être fixé au départ sur l'ADN endommagé ou introduit dans celui-ci lors du processus de réparation.In this method, the damaged DNA of which at least one lesion is known to be fixed on a solid support such as a biochip, then it is subjected to the action of a protein such as a repair enzyme, or '' a composition which could contain this protein, and the repair is followed by means of a marker which can be initially fixed on the DNA damaged or introduced into it during the repair process.
Ainsi, le procédé peut servir à caractériser les protéines impliquées dans la réparation de l'ADN. Il peut aussi être utilisé pour mettre en évidence la non-fonctionalité de certaines protéines vis-à-vis de lésions connues, ou encore' pour détecter l'absence de protéines de réparation de l'ADN dans des compositions qui normalement devraient en contenir.Thus, the method can be used to characterize the proteins involved in DNA repair. It can also be used to demonstrate the non-functionality of certain proteins with respect to known lesions, or even to detect the absence of DNA repair proteins in compositions which normally should contain them.
L'obtention de ces résultats peut être utile pour des applications en diagnostic. En effet, dans certaines maladies, des gènes de la réparation sont mutés et 1 ' activité enzymatique ou la protéine associée ne sont pas fonctionnelles ou le sont partiellement (xeroderma pigmentosum par exemple) .Obtaining these results can be useful for diagnostic applications. Indeed, in certain diseases, repair genes are mutated and the enzymatic activity or the associated protein are not functional or are partially functional (xeroderma pigmentosum for example).
Dans ce procédé le support solide comprend au moins un site de fixation défini d'un fragment d'ADN endommagé et avantageusement plusieurs sites de fixation permettant l'immobilisation de différents fragments d'ADN endommagés préalablement choisis.In this method, the solid support comprises at least one defined binding site for a damaged DNA fragment and advantageously several binding sites allowing the immobilization of different previously selected damaged DNA fragments.
Les protéines dont on veut mesurer l'activité sont les protéines impliquées dans le processus de réparation qui comporte généralement une reconnaissance du dommage, une incision de la chaîne d'ADN, une excision du dommage ou de fragments d'acides nucléiques, une synthèse in situ de l'ADN, et une ligation du brin néoformé.The proteins whose activity we want to measure are the proteins involved in the repair process which generally involves recognition of the damage, an incision in the DNA chain, excision of the damage or fragments of nucleic acids, an in synthesis situ of DNA, and a ligation of the newly formed strand.
A titre d'exemple, les protéines impliquées dans ce processus peuvent être choisies parmi : les protéines impliquées dans la reconnaissance des lésions telles que XPA, le facteur de transcription TFIIH et les polypeptides qui le composent, XPC, XPF, XPG et leurs protéines associées (famille ERCC, etc) ) , HSSB, etc (voir Sancar, A. (1995) Annu. Rev. Genetics, 29, pages 69-105, [10]), les protéines impliquées dans l'excision des lésions telles que les glycosylases, - les protéines impliqués dans la resynthèse du (des) nucléotide ( ) s du brin excisé telles que les polymérases, et les protéines impliquées dans la ligation de brins néoformés telles que les ligases. Selon un premier mode de réalisation du procédé de l'invention, le marqueur est présent sur l'ADN endommagé fixé sur le support et il est éliminé par l'action de la protéine dans l'étape b) .For example, the proteins involved in this process can be chosen from: the proteins involved in the recognition of lesions such as XPA, the transcription factor TFIIH and the polypeptides which compose it, XPC, XPF, XPG and their associated proteins (ERCC family, etc.), HSSB, etc. (see Sancar, A. (1995) Annu. Rev. Genetics, 29, pages 69-105, [10]), proteins involved in the excision of lesions such as glycosylases, - proteins involved in the resynthesis of the nucleotide (s) excised strand such as polymerases, and proteins involved in the ligation of newly formed strands such as ligases. According to a first embodiment of the method of the invention, the marker is present on the damaged DNA fixed on the support and it is eliminated by the action of the protein in step b).
Ce mode de réalisation peut être utilisé par exemple pour déterminer l'activité d'enzymes d'incision ou d'excision des lésions d'ADN endommagé. Dans ce cas, le marqueur peut être présent sur une extrémité de l'ADN endommagé. Ainsi, une incision ou une excision provoque l'élimination du fragment d'ADN endommagé portant le marqueur et la perte du signal sur le support à l'endroit où est fixé l'ADN endommagé.This embodiment can be used for example to determine the activity of enzymes for incision or excision of damaged DNA lesions. In this case, the marker may be present on one end of the damaged DNA. Thus, an incision or an excision causes the elimination of the damaged DNA fragment carrying the marker and the loss of the signal on the support at the place where the damaged DNA is fixed.
Le premier mode de réalisation peut également être mis en œuvre avec un marqueur spécifique des lésions de l'ADN endommagé tel qu'un anticorps, qui révèle ces lésions avant l'étape b) . Après action de la protéine et réparation des lésions, ce marqueur ne peut se fixer et on peut observer une perte du signal du marqueur représentative de l'activité de la protéine de réparation.The first embodiment can also be implemented with a specific marker for damaged DNA lesions such as an antibody, which reveals these lesions before step b). After action of the protein and repair of lesions, this marker cannot bind and a loss of the marker signal representative of the activity of the repair protein can be observed.
Ainsi, le rapport du signal émis par les anticorps spécifiques avant la réparation de l'ADN sur le signal émis par les anticorps après réaction de la protéine, par exemple réaction enzymatique, est corrélé au taux de réparation de l'ADN. Dans ce dernier cas, la réparation du dommage est évaluée par la disparition d'un signal spécifique du dommage.Thus, the ratio of the signal emitted by the specific antibodies before the DNA repair to the signal emitted by the antibodies after reaction of the protein, for example enzymatic reaction, is correlated with the rate of DNA repair. In the latter case, compensation for damage is assessed by the disappearance of a specific damage signal.
Selon un second mode de réalisation du procédé de l'invention, le marqueur est présent dans la composition de réparation et est introduit dans l'ADN fixé sur le support pendant l'étape b) . Dans ce cas, on peut par exemple ajouter dans la composition de réparation, des nucleotides modifiés incorporés par les polymérases et pouvant servir au marquage du brin néoformé. Ces nucleotides modifiés peuvent porter une biotine, un haptène, un composé fluorescent ou toute autre molécule compatible avec le marquage des acides nucléiques. Ces marqueurs introduits lors de 1 ' étape de resynthèse peuvent être ensuite détectés et le signal correspondant corrélé à un taux de réparation. Les marqueurs utilisables pour déterminer l'activité des protéines impliquées dans la réparation de l'ADN, peuvent être de différents types du moment qu'ils émettent un signal détectable ou puissent être révélés en émettant un signal détectable. Plusieurs marqueurs ou mode de révélation peuvent être utilisés simultanément ou de façon séquentielle de façon à mettre en évidence des changements d'état intervenus sur les fragments d'ADN endommagés, par les activités proteiques liées à la réparation. Le marqueur peut être en particulier une molécule d'affinité, un composé fluorescent, un anticorps, un haptène ou une biotine.According to a second embodiment of the method of the invention, the marker is present in the repair composition and is introduced into the DNA fixed on the support during step b). In this case, it is possible, for example, to add to the repair composition, modified nucleotides incorporated by the polymerases and which can be used for labeling the newly formed strand. These modified nucleotides can carry a biotin, a hapten, a fluorescent compound or any other molecule compatible with the labeling of nucleic acids. These markers introduced during the resynthesis step can then be detected and the corresponding signal correlated to a repair rate. The markers that can be used to determine the activity of proteins involved in DNA repair can be of different types as long as they emit a detectable signal or can be revealed by emitting a detectable signal. Several markers or mode of development can be used simultaneously or in a way sequential so as to highlight changes in state which have occurred in damaged DNA fragments, by the protein activities linked to repair. The marker can in particular be an affinity molecule, a fluorescent compound, an antibody, a hapten or a biotin.
De préférence, selon l'invention, on utilise comme marqueur ou révélateur du marqueur, des composés fluorescents à fluorescence directe ou à fluorescence indirecte. De telles molécules peuvent être par exemple l'avidine, révélée par la streptavidine-phycoérithrine, des cryptâtes d'europium, des composés fluorescents tels que la fluorescéines , la rhodamine, etc...Preferably, according to the invention, use is made, as marker or marker revealer, of fluorescent compounds with direct fluorescence or with indirect fluorescence. Such molecules may for example be avidin, revealed by streptavidin-phycoerithrin, europium cryptates, fluorescent compounds such as fluoresceins, rhodamine, etc.
Les propriétés de transfert d'énergie entre molécules fluorescentes peuvent être également exploitées pour quantifier une activité enzymatique impliquée dans la réparation sur un oligonucléotide substrat. Dans le cas où 1 ' oligonucléotide comportant la ou les bases lésées, porte également une molécule fluorescente autorisant un transfert d'énergie avec une autre molécule fluorescente située soit sur le même oligonucléotide, soit sur un oligonucléotide en contact avec le premier, l'émission d'un signal après excitation prouve la présence de la lésion sur le support. L'incision de 1 ' oligonucléotide comportant la ou les lésions par une enzyme de réparation conduit à l'élimination du fragment incisé et à la perte du signal fluorescent, le transfert d'énergie ne pouvant avoir lieu. Ce changement de signal peut être mesuré et corrélé avec un taux de digestion spécifique.The energy transfer properties between fluorescent molecules can also be exploited to quantify an enzymatic activity involved in repair on a substrate oligonucleotide. In the case where the oligonucleotide comprising the damaged base (s), also carries a fluorescent molecule authorizing an energy transfer with another fluorescent molecule located either on the same oligonucleotide or on an oligonucleotide in contact with the first, the emission of a signal after excitation proves the presence of the lesion on the support. The incision of the oligonucleotide comprising the lesion (s) by a repairing enzyme leads to the elimination of the incised fragment and to the loss of the fluorescent signal, the energy transfer being unable to take place. This change in signal can be measured and correlated with a specific digestion rate.
Selon l'invention, la fixation de l'ADN endommagé sur le support solide peut être effectuée par des procédés classiques.According to the invention, the fixing of the damaged DNA to the solid support can be carried out by conventional methods.
Ainsi, on peut synthétiser directement sur le support solide un ADN endommagé au moyen de synthétiseurs automatiques permettant par exemple l'incorporation de bases modifiées. On peut aussi fixer l'ADN endommagé sur le support solide par toute méthode compatible avec le maintien de l'intégrité du fragment d'ADN contenant les dommages, par exemple par hybridation avec un autre fragment d'ADN immobilisé sur le support, par immobilisation par une molécule d'affinité, ou par fixation directe sur la puce par dépôt ou tout autre moyen .Thus, damaged DNA can be synthesized directly on the solid support by means of automatic synthesizers allowing for example the incorporation of modified bases. The damaged DNA can also be fixed on the solid support by any method compatible with maintaining the integrity of the DNA fragment containing the damage, for example by hybridization with another DNA fragment immobilized on the support, by immobilization by an affinity molecule, or by direct attachment to the chip by deposition or any other means.
De préférence, on utilise comme support solide une biopuce sur laquelle sont fixés, à des endroits déterminés, des fragments d'ADN comportant des lésions, identiques ou différentes, parfaitement identifiés, ou comportant des lésions multiples non parfaitement identifiées.Preferably, a biochip is used as solid support on which are fixed, at determined locations, DNA fragments comprising lesions, identical or different, perfectly identified, or comprising multiple lesions which are not perfectly identified.
Les fragments d'ADN comportant les lésions et immobilisés peuvent être de courts oligonucléotidesThe DNA fragments comprising the lesions and immobilized can be short oligonucleotides
(15-100 bases de long, de préférence 15 à 50 bases de long) ou des fragments plus longs (100-20 000 bases) . Différentes méthodes existent rendant possible l'incorporation d 'oligonucléotides comportant des lésions dans de longs fragments d'ADN : Réaction de(15-100 bases long, preferably 15 to 50 bases long) or longer fragments (100-20,000 bases). Different methods exist making it possible to incorporate oligonucleotides comprising lesions in long DNA fragments:
Polymérisation en Chaîne (PCR) , ligation. Les fragments d'ADN immobilisés peuvent être sous forme de simple ou de double brin. La fixation de l'ADN modifié peut se faire directement ou via une molécule intermédiaire (type biotine, antigène, acide nucléique, etc.). Les longs fragments d'ADN peuvent être également endommagés par tout traitement physique ou chimique induisant des lésions. Par exemple, ils peuvent être irradiés par rayonnement radioactif, solaire ou ultraviolet. Ils peuvent être endommagés par des réactions de photosensibilisation, par des agents chimiques, par des carcinogènes . Dans ce cas, les lésions seront induites statistiquement le long de la chaîne nucléotidique . La qualité, la spécificité et la quantité des lésions induites dépend du choix de l'agent chimique ou physique provoquant les lésions. On peut ainsi cibler la catégorie de lésions que l'on souhaite introduire dans un fragment d'ADN.Chain polymerization (PCR), ligation. The fragments immobilized DNA can be in the form of single or double strand. The binding of the modified DNA can be done directly or via an intermediate molecule (biotin type, antigen, nucleic acid, etc.). Long DNA fragments can also be damaged by any physical or chemical treatment that induces lesions. For example, they can be irradiated by radioactive, solar or ultraviolet radiation. They can be damaged by photosensitization reactions, by chemical agents, by carcinogens. In this case, the lesions will be induced statistically along the nucleotide chain. The quality, specificity and quantity of the lesions induced depends on the choice of the chemical or physical agent causing the lesions. We can thus target the category of lesions that we wish to introduce into a DNA fragment.
Le support peut être par exemple une biopuce comportant des fragments d'ADN, sur laquelle on fixe par hybridation l'ADN endommagé au moyen d'un oligonucléotide comportant une partie complémentaire de l ' ADN endommagé et une partie complémentaire de l'un des fragments d'ADN de la biopuce.The support can for example be a biochip comprising DNA fragments, on which the damaged DNA is fixed by hybridization by means of an oligonucleotide comprising a complementary part of the damaged DNA and a complementary part of one of the fragments DNA from the biochip.
L'invention bénéficie entre autre, des avantages de la synthèse chimique d' oligonucléotides et donc du fait que l'on peut choisir exactement les séquences contenant et environnant le dommage.The invention benefits inter alia from the advantages of the chemical synthesis of oligonucleotides and therefore from the fact that one can choose exactly the sequences containing and surrounding the damage.
Le même support peut être fonctionnalisé par des fragments nucléotidiques de séquences identiques ou différentes. Les dommages peuvent être localisés à divers endroits définis ou non des séquences .The same support can be functionalized by nucleotide fragments of identical or different sequences. Damage can be located at various locations, defined or not, of the sequences.
La nature des fragments fixés sur le support (taille, nombre de dommages, emplacement des dommages ... ) est modulable et dépend de la nature des informations recherchées .The nature of the fragments fixed on the support (size, number of damages, location of damages, etc.) is flexible and depends on the nature of the information sought.
Les fragments d'ADN comportant les lésions ou bien les fragments complémentaires des fragments comportant les lésions peuvent également comporter des marqueurs utiles pour la détection des transformations de l'ADN liées à une activité enzymatique de réparation. Ces marqueurs peuvent être des molécules d'affinité, des composés fluorescents, ou tout système de détection spécifique lié à un type de puce spécifique.The DNA fragments comprising the lesions or else the complementary fragments of the fragments comprising the lesions can also contain markers useful for the detection of DNA transformations linked to an enzymatic repair activity. These markers can be affinity molecules, fluorescent compounds, or any specific detection system linked to a specific type of chip.
Lorsque les fragments d'ADN sont immobilisés sur la puce (puce de référence) , une révélation est réalisée permettant de caractériser les états dans lesquels se trouvent les fragments. Les différents plots de la puce sont ainsi caractérisés par les signaux qu'ils émettent. Ils servent de signal de référence.When the DNA fragments are immobilized on the chip (reference chip), a revelation is carried out making it possible to characterize the states in which the fragments are found. The different pads of the chip are thus characterized by the signals they emit. They serve as a reference signal.
Ensuite, la puce de référence ou bien une puce comportant des fragments d'ADN endommagés identiques à ceux de la puce de référence, dite puce de test, est incubée en présence de solutions susceptibles de contenir les activités enzymatiques de réparation. Les fragments d'ADN modifiés fixés sur la puce sont susceptibles d'être transformés par les activités enzymatiques présentes.Then, the reference chip or else a chip comprising damaged DNA fragments identical to those of the reference chip, known as the test chip, is incubated in the presence of solutions capable of containing the enzymatic repair activities. The modified DNA fragments attached to the chip are susceptible to transformation by the enzymatic activities present.
Une deuxième lecture de la puce de test ou de la puce de référence est réalisée dans des conditions appropriées et après les étapes de révélation nécessaires. Les plots de la puce peuvent ainsi émettre des signaux différents de la première lecture, signifiant qu'une ou plusieurs réactions enzymatiques ont eu lieu sur la puce.A second reading of the test chip or of the reference chip is carried out under appropriate conditions and after the necessary disclosure steps. The studs of the chip can thus emit signals different from the first reading, signifying that one or more enzymatic reactions have taken place on the chip.
Il est à noter que la mesure de la transformation des ADN endommagés par les enzymes liées aux systèmes de réparation peut être suivie par enregistrement continu des signaux émis par les différents plots de la puce, si les marqueurs utilisés sont compatibles avec un tel système de détection.It should be noted that the measurement of the transformation of DNA damaged by enzymes linked to repair systems can be followed by continuous recording of the signals emitted by the various studs of the chip, if the markers used are compatible with such a detection system. .
Le procédé de l'invention peut être utilisé pour l'étude d'enzymes ou de facteurs proteiques purifiés, ainsi que pour l'étude d'activités présentes dans des extraits cellulaires .The method of the invention can be used for the study of enzymes or purified protein factors, as well as for the study of activities present in cellular extracts.
Il peut être utilisé également pour étudier les spécificités de différentes lésions de l'ADN pour certaines enzymes de réparation, en particulier pour les glycosylases. Dans le même domaine, on peut suivre les cinétiques de réparation de ces lésions en fonction de différents facteurs. Ces facteurs peuvent être liés à la séquence nucléotidique (effet des bases environnantes sur la réparation par exemple) , à des inhibiteurs ou au contraire à des stimulateurs d'activités réparatrices.It can also be used to study the specificities of different DNA lesions for certain repair enzymes, in particular for glycosylases. In the same field, we can follow the kinetics of repair of these lesions according to different factors. These factors can be linked to the nucleotide sequence (effect of the surrounding bases on repair for example), to inhibitors or on the contrary to stimulators of repairing activities.
Le suivi de la réparation d'une lésion identifiée est intéressant pour le dosage d'activités enzymatiques impliquées dans les étapes de reconnaissance et d'excision d'une lésion particulière.Monitoring the repair of an identified lesion is interesting for the determination of the enzymatic activities involved in the stages of recognition and excision of a particular lesion.
Par exemple, on sait qu'il existe des glycosylases spécifiques de l'excision d'une lésion particulière. Une déficience d'une enzyme particulière conduit donc à la non réparation d'une lésion particulière.For example, we know that there are glycosylases specific to excision of a particular lesion. A deficiency of a particular enzyme therefore leads to the non-repair of a particular lesion.
La miniaturisation du support permet d'améliorer la sensibilité du système en particulier lors de l'utilisation d'extraits cellulaires. L'invention facilite la détection des différentes protéines liées aux systèmes de réparation à partir de cellules en culture ou de biopsies. Par ailleurs, en utilisant une biopuce comportant plusieurs séquences d'ADN endommagé présentant des lésions différentes connues, on peut détecter les capacités de réparation de ces différents dommages pour une quantité limitée d'un même extrait cellulaire. Ceci est très important, notamment dans le cas de diagnostic de certaines pathologies et maladies génétiques .The miniaturization of the support improves the sensitivity of the system, in particular when using cell extracts. The invention facilitates the detection of the various proteins linked to repair systems from cultured cells or from biopsies. Furthermore, by using a biochip comprising several damaged DNA sequences presenting different known lesions, it is possible to detect the repair capacities of these different damages for a limited quantity of the same cell extract. This is very important, especially in the case of diagnosis of certain genetic pathologies and diseases.
L'invention présente notamment de nombreux avantages .The invention has in particular many advantages.
En effet, elle permet la miniaturisation des systèmes de dosage d'activités enzymatiques, et plus particulièrement ceux liés à la réparation. Il en résulte un gain de sensibilité. Cette amélioration est très importante puisque les extraits cellulaires sont difficiles à obtenir, en particulier lorsqu'ils proviennent de prélèvements biologiques et non pas de lignées cellulaires.In fact, it allows the miniaturization of systems for assaying enzymatic activities, and more particularly those linked to repair. This results in a gain in sensitivity. This improvement is very important since cell extracts are difficult to obtain, in particular when they come from biological samples and not from cell lines.
Elle permet d'obtenir des informations précises sur la fonctionnalité, l'induction et la répression des différentes étapes liées à la réparation des dommages de l'ADN dans des systèmes procaryotes ou eucaryotes . Elle permet ainsi d'accéder plus facilement à des dosages biologiques d'activités enzymatiques chez l'homme. En particulier, elle permet la détection de déficiences enzymatiques telles qu'on les trouve dans des pathologies faisant intervenir la radiosensibilité ou la photosensibilité comme le Xeroderma pigmentosum, la maladie de Cockaine . Elle permet d'étudier plus facilement les effets du vieillissement sur les activités enzymatiques de réparation. L'invention permet aussi d'étudier le comportement d'enzymes simultanément envers différentes lésions incorporées dans des fragments nucléotidiques immobilisés sur le même support miniaturisé. Ceci facilite la comparaison des résultats entre eux et les rend plus fiables et reproductibles. Il en résulte également un gain de temps important .It provides precise information on the functionality, induction and repression of the various stages linked to the repair of DNA damage in prokaryotic or eukaryotic systems. It thus allows easier access to biological assays of enzymatic activities in humans. In particular, it allows the detection of enzymatic deficiencies as found in pathologies involving radiosensitivity or photosensitivity such as Xeroderma pigmentosum, Cockaine disease. It makes it easier to study the effects of aging on enzymatic repair activities. The invention also makes it possible to study the behavior of enzymes simultaneously towards different lesions incorporated in nucleotide fragments immobilized on the same miniaturized support. This makes it easier to compare results and make them more reliable and reproducible. This also results in significant time savings.
L'invention apporte pour la première fois une approche globale de 1 ' évaluation de la fonctionnalité des systèmes de réparation avec accès à plusieurs lésions et plusieurs mécanismes ou étapes de réparation simultanément.The invention provides for the first time a global approach to the evaluation of the functionality of repair systems with access to several lesions and several repair mechanisms or stages simultaneously.
Une analyse directe des signaux émis par les différents plots de la puce après action des systèmes enzymatiques à étudier, permet de remonter directement aux étapes des différents systèmes de réparation et de conclure sur leur fonctionnement.A direct analysis of the signals emitted by the various studs on the chip after the action of the enzymatic systems to be studied makes it possible to go back directly to the stages of the various repair systems and to conclude on their operation.
L'invention permet le dosage d'une protéine particulière impliquée dans la réparation d'une lésion ciblée, comme par exemple le dosage d'une glycosylase impliquée dans l'excision des bases oxydées. Dans ce cas, la lésion connue pour être le substrat préférentiel de la glycosylase ciblée sera incorporée dans un oligonucléotide de synthèse et fixée sur la puce à un endroit déterminé. On voit donc que l'on peut, en fixant plusieurs oligonucléotides comportant des lésions ciblées et différentes en des endroits différents de la puce, avoir une évaluation de la fonctionnalité des différentes glycosylases impliquées dans le système de REB. Mais, l'invention permet également le dosage de protéines impliquées dans des étapes communes à la réparation de toutes les lésions comme les polymérases et les ligases. Dans ce dernier cas on peut utiliser des fragments d'ADN comportant des lésions moins précisément identifiées.The invention allows the determination of a particular protein involved in the repair of a targeted lesion, such as the determination of a glycosylase involved in the excision of oxidized bases. In this case, the lesion known to be the substrate preferential of the targeted glycosylase will be incorporated into a synthetic oligonucleotide and fixed on the chip at a determined location. It can therefore be seen that, by fixing several oligonucleotides comprising targeted and different lesions at different locations on the chip, we can have an evaluation of the functionality of the different glycosylases involved in the REB system. However, the invention also allows the assay of proteins involved in steps common to the repair of all lesions such as polymerases and ligases. In the latter case, it is possible to use DNA fragments comprising lesions which are less precisely identified.
L'invention se caractérise également par le fait que le choix des différents substrats fixés en des plots différents de la puce permet de cibler le système de réparation auquel on s'intéresse. En effet, la fixation d' oligonucléotides courts (inférieurs à 25 bases) comportant des lésions permet de cibler les protéines du système de REB. La fixation de fragments plus longs (supérieurs à 50 bases) comportant les lésions permet de cibler à la fois les protéines du système REB et REN. La sélection précise des dommages fixés permet également de cibler le systèmes de réparation à étudier.The invention is also characterized by the fact that the choice of the different substrates fixed in different pads of the chip makes it possible to target the repair system in which one is interested. Indeed, the fixation of short oligonucleotides (less than 25 bases) comprising lesions makes it possible to target the proteins of the REB system. The fixation of longer fragments (greater than 50 bases) comprising the lesions makes it possible to target both the proteins of the REB system and REN. The precise selection of fixed damages also makes it possible to target the repair systems to be studied.
Ainsi, on peut utiliser le procédé de l'invention pour l'étude de la spécificité et la fonctionnalité des protéines impliquées dans la réparation vis-à-vis de lésions connues d'ADN, pour suivre la cinétique de réparation de lésions de l'ADN par des protéines, pour évaluer l'effet génotoxique de substances ou d'agents physiques inhibant ou stimulant la synthèse des protéines' impliquées dans la réparation de l'ADN, ou pour le diagnostic de déficiences des protéines de la réparation liées à des maladies. D'autres caractéristiques et avantages de l'invention apparaîtront mieux à la lecture des exemples suivants donnés bien entendu à titre illustratif et non limitatif en référence aux dessins annexés .Thus, the method of the invention can be used for the study of the specificity and the functionality of the proteins involved in repair with respect to known DNA lesions, to follow the kinetics of repair of lesions of the DNA by proteins, to assess the genotoxic effect of substances or physical agents inhibiting or stimulating the synthesis of proteins' involved in DNA repair, or for the diagnosis of deficiencies in repair proteins linked to diseases. Other characteristics and advantages of the invention will appear better on reading the following examples given, of course, by way of illustration and not limitation with reference to the appended drawings.
Brève description des dessinsBrief description of the drawings
La figure 1 représente schématiquement l'état d'un système dans la première étape du procédé de l'invention.FIG. 1 schematically represents the state of a system in the first step of the method of the invention.
La figure 2 représente schématiquement l'état du système de la figure 1 après l'étape b) du procédé de l'invention.FIG. 2 schematically represents the state of the system of FIG. 1 after step b) of the method of the invention.
Description détaillée des modes de réalisationDetailed description of the embodiments
Exemple 1Example 1
Dans cet exemple, on utilise le premier mode de réalisation du procédé de l'invention pour déterminer une activité de réparation de type glycosylase sur un oligonucléotide endommagé comportant comme lésion une 8-oxo-7,8 dihydro-2 ' -désoxyguanosine .In this example, the first embodiment of the method of the invention is used to determine a glycosylase-type repair activity on a damaged oligonucleotide comprising as lesion an 8-oxo-7,8 dihydro-2 '-deoxyguanosine.
On utilise une biopuce de type MICAM pb4 obtenue comme il est décrit par Livache et al, Biosens.Using a MICAM pb4 biochip obtained as described by Livache et al, Biosens.
Bioelectron, 13, 1998, page 629 et 634 [8], comportant quatre plots fonctionnalisés par quatre oligonucléotides de synthèse de séquences différentes (H, I, J, K) . La séquence H est la suivante :Bioelectron, 13, 1998, page 629 and 634 [8], comprising four studs functionalized by four synthetic oligonucleotides of different sequences (H, I, J, K). The H sequence is as follows:
Séquence H : 5' TTTTT CCA CAC GGT AGG TAT CAG TCSequence H: 5 'TTTTT CCA CAC GGT AGG TAT CAG TC
La partie fonctionnalisée de la biopuce est incubée en présence d'un hybride formé de 1 ' oligonucléotide (O) endommagé, comportant une 8-oxo- 7 , 8-dihydro-2 ' -désoxyguanosine, et d'un oligonucléotide (cOcH) comportant une partie complémentaire de 1 Oligonucléotide O (cO) et une partie complémentaire de l' oligonucléotide H fixé sur la puce (cH) . L 'oligonucléotide O comporte une biotine à son extrémité 3 ' .The functionalized part of the biochip is incubated in the presence of a hybrid formed from one damaged oligonucleotide (O), comprising an 8-oxo-7, 8-dihydro-2 '- deoxyguanosine, and an oligonucleotide (cOcH) comprising a complementary part of 1 Oligonucleotide O (cO) and a complementary part of oligonucleotide H fixed on the chip (cH). The oligonucleotide O has a biotin at its 3 'end.
0 : 5'GAA CTA GTG XAT CCC CCG GGC TGC - Biotine 3' (X étant la 8-oxo-7,8 dihydro-2 ' -désoxyguanosine) .0: 5'GAA CTA GTG XAT CCC CCG GGC TGC - Biotin 3 '(X being 8-oxo-7,8 dihydro-2'-deoxyguanosine).
cOcH : 5 ' GCA GCC CGG GGG ATC CAC TAG TTC GAC TGA CTA CCG TGT GGcOcH: 5 'GCA GCC CGG GGG ATC CAC TAG TTC GAC TGA CTA CCG TGT GG
L'hybride O-cOcH est obtenu par incubation pendant 60 min à37°C de 10 pmoles de cOcH et 12 pmoles de O dans 200 μl de tampon PBS contenant 0,2 M de NaCl . 20 μl de cette solution sont prélevés et ajoutés sur la partie fonctionnalisée de la puce qui forme une cuvette. Après 1 heure d'incubation en milieu humide à 37°C, la puce est trempée dans un tampon de lavage (PBS/NaCl 0,2M/tween 20 0,05 %) .The O-cOcH hybrid is obtained by incubation for 60 min at 37 ° C of 10 pmol of cOcH and 12 pmol of O in 200 μl of PBS buffer containing 0.2 M NaCl. 20 μl of this solution are removed and added to the functionalized part of the chip which forms a cuvette. After 1 hour incubation in a humid environment at 37 ° C., the chip is soaked in a washing buffer (PBS / 0.2 M NaCl / 0.05% tween 20).
La figure 1 représente le système obtenu par fixation de l' oligonucléotide O sur le plot 1 de la biopuce comportant l' oligonucléotide H. Cet oligonucléotide H est hybride avec la séquence cH de 1 ' oligonucléotide 3 qui comporte également une séquence hybridée avec 1 ' oligonucléotide endommagé O portant une biotine 5 à son extrémité 3 ' et une lésion 7.FIG. 1 represents the system obtained by fixing the oligonucleotide O on the pad 1 of the biochip comprising the oligonucleotide H. This oligonucleotide H is hybridized with the sequence cH of The oligonucleotide 3 which also comprises a sequence hybridized with the damaged oligonucleotide O carrying a biotin 5 at its 3 'end and a lesion 7.
L ' oligonucléotide O comportant la lésion et fixé sur la biopuce par hybridation, est révélé après incubation pendant 10 min à température ambiante avec 20 μl de tampon PBS/NaCl contenant 0,5 % de sérum albumine bovine BSA et 1 μl de streptavidine- phycoérythrine R (0,5 mg/ml ; Jackson ImmunoResearch) . Après lavage dans PBS/NaCl/t een, la biopuce est observée sous microscope à fluorescence et le signal est analysé avec le logiciel Image Pro Plus.The oligonucleotide O containing the lesion and fixed on the biochip by hybridization, is revealed after incubation for 10 min at room temperature with 20 μl of PBS / NaCl buffer containing 0.5% of bovine serum albumin BSA and 1 μl of streptavidin-phycoerythrin R (0.5 mg / ml; Jackson ImmunoResearch). After washing in PBS / NaCl / t een, the biochip is observed under a fluorescence microscope and the signal is analyzed with Image Pro Plus software.
Le signal est intégré en pixels. Les plots fonctionnalisés émettent un léger signal fluorescent même en l'absence de toute hybridation. Les plots non fonctionnalisés sont noirs.The signal is integrated in pixels. The functionalized studs emit a slight fluorescent signal even in the absence of any hybridization. The non-functionalized studs are black.
On observe sur le plot fonctionnalisé par 1 ' oligonucléotide H un signal fluorescent intense saturant à 250 pixels. Pour comparaison le plot fonctionnalisé par la séquence I, présente une fluorescence maximale d'environ 110 pixels. Le rapport signal sur bruit (H/I) est de 2,27.An intense fluorescent signal saturating at 250 pixels is observed on the pad functionalized with the oligonucleotide H. For comparison, the plot functionalized by sequence I has a maximum fluorescence of around 110 pixels. The signal to noise ratio (H / I) is 2.27.
L'hybridation de 1 ' oligonucléotide comportant une lésion de l'ADN est donc spécifique. De plus, on voit qu'il est possible de fixer un fragment d'acide nucléique comportant un dommage donné à un site prédéfini sur une biopuce.Hybridization of one oligonucleotide with DNA damage is therefore specific. In addition, it is seen that it is possible to fix a nucleic acid fragment comprising a damage given to a predefined site on a biochip.
La biopuce est lavée avec NaOH 0,1 N pendant 10 min, puis rincée 10 min avec H20, ce qui a pour effet d'éliminer les oligonucléotides 0 et cOcH de la biopuce. On vérifie au microscope que tout signal a disparu .The biochip is washed with 0.1 N NaOH for 10 min, then rinsed for 10 min with H 2 0, which has the effect of removing oligonucleotides 0 and cOcH from the biochip. We check under the microscope that any signal has faded away .
La puce est à nouveau hybridée avec l'hybride O-cOcH dans les conditions définies précédemment. Après lavage au PBS/NaCl/tween, la puce est équilibrée 10 min avec un tampon RCl 0,1 M, Tris, puis 20 μl de ce même tampon contenant 0,5 μg de Fapy DNA Glycosylase (S. Boiteux, CEA Fontenay-aux-Roses, France) sont ajoutés. La puce est incubée 30 min à 37°C en atmosphère humide . Après lavage en PBS/NaCl/tween, on procède comme précédemment à 1 ' étape de révélation avec la streptavidine-phycoérythrine R. Le signal est enregistré après lavage au PBS/tween. La fluorescence intense sur le plot H a disparu. Le rapport signal sur bruit (H/I) est de 1,04.The chip is again hybridized with the O-cOcH hybrid under the conditions defined above. After washing with PBS / NaCl / tween, the chip is balanced for 10 min with a 0.1 M RCl buffer, Tris, then 20 μl of this same buffer containing 0.5 μg of Fapy DNA Glycosylase (S. Boiteux, CEA Fontenay- aux-Roses, France) are added. The chip is incubated for 30 min at 37 ° C. in a humid atmosphere. After washing with PBS / NaCl / tween, the revelation step is carried out as above with the streptavidin-phycoerythrin R. The signal is recorded after washing with PBS / tween. The intense fluorescence on the H stud has disappeared. The signal to noise ratio (H / I) is 1.04.
La figure 2 représente l'état du système après réaction avec l'enzyme de type glycosylase.FIG. 2 represents the state of the system after reaction with the glycosylase type enzyme.
Sur cette figure, on voit que 1 ' oligonucléotide 0 a été éliminé de la biopuce. En effet, l'enzyme Fapy DNA glycosylase a coupé 1 ' oligonucléotide O au niveau de la 8-oxo-7 , 8-dihydro- désoxyguanosine. Les courts fragments d'ADN ainsi engendrés forment des hybrides instables dans les conditions utilisées et sont éliminés de la biopuce. Ceci entraîne la disparition du signal fluorescent dû à la révélation de la biotine.In this figure, it can be seen that the oligonucleotide 0 has been eliminated from the biochip. Indeed, the enzyme Fapy DNA glycosylase cut the oligonucleotide O at the level of 8-oxo-7, 8-dihydro-deoxyguanosine. The short DNA fragments thus generated form hybrids which are unstable under the conditions used and are eliminated from the biochip. This results in the disappearance of the fluorescent signal due to the revelation of biotin.
Cet exemple montre ainsi qu'une activité de réparation de l'ADN est détectable sur un microsupport portant un fragment d'ADN comportant une lésion d'une base de l'ADN. Exemple 2This example thus shows that a DNA repair activity is detectable on a microcarrier carrying a DNA fragment comprising a lesion of a DNA base. Example 2
Cet exemple illustre la détection d'une activité de type glycosylase dans un lysat cellulaire en utilisant le premier mode de réalisation du procédé de l'invention.This example illustrates the detection of a glycosylase type activity in a cell lysate using the first embodiment of the method of the invention.
Dans cet exemple, 1 ' oligonucléotide O utilisé dans l'exemple 1, comportant la 8-oxo-7,8- dihydro-2 ' -désoxynucléotide, est hybride comme dans l'exemple 1 sur la biopuce pb4, ce qui correspond au système représenté sur la figure 1.In this example, the oligonucleotide O used in example 1, comprising the 8-oxo-7,8-dihydro-2 '- deoxynucleotide, is hybridized as in example 1 on the biochip pb4, which corresponds to the system shown in figure 1.
Un lysat cellulaire total est préparé à partir de cellules Hela en culture.A total cell lysate is prepared from Hela cells in culture.
Les cellules sont trypsinées puis lavées dans un tampon PBS . Le culot contenant environ 15.10 cellules est repris par 1 ml de tampon de lyseThe cells are trypsinized and then washed in PBS buffer. The pellet containing approximately 15.10 cells is taken up in 1 ml of lysis buffer
(Tris-HCl 10 mM pH 7,5, MgCl2 10 mM, KCl 10 mM, EDTA 1 mM, contenant 1 pastille pour 10 ml d' antiproteases(10 mM Tris-HCl pH 7.5, 10 mM MgCl 2, 10 mM KCl, 1 mM EDTA, containing 1 tablet per 10 ml of antiproteases
«Complète, Mini» (Boehringer Mannheim) et 5 μl de"Complete, Mini" (Boehringer Mannheim) and 5 μl of
Phénylméthylsulphonyl Fluoride (PMSF, Sigma, 17,4 mg/ l dans 1 ' isopropanol) . Les cellules sont broyées, puis le lysat est centrifugé à 4°C, pendant 50 min àPhenylmethylsulphonyl Fluoride (PMSF, Sigma, 17.4 mg / l in isopropanol). The cells are ground, then the lysate is centrifuged at 4 ° C, for 50 min at
65 000 tours/min dans une ultracentrifugeuse Beckman.65,000 rpm in a Beckman ultracentrifuge.
Le surnageant est récupéré puis on rajoute 200 μl de glycérol et 20 μl de dithiotréitol 0,1M. Les lysats sont aliquotés et stockés à -80°C.The supernatant is recovered and then 200 μl of glycerol and 20 μl of 0.1M dithiotreitol are added. The lysates are aliquoted and stored at -80 ° C.
La biopuce est ensuite incubée avec 20 μl d'extrait cellulaire pendant 45 min à 37°C. La révélation est effectuée comme dans l'exemple 1.The biochip is then incubated with 20 μl of cell extract for 45 min at 37 ° C. The revelation is carried out as in Example 1.
Le signal du plot H est enregistré. Il est de 180 pixels.The signal from pad H is recorded. It is 180 pixels.
L'expérience est renouvelée avec du lysat dénaturé (chauffage à 100°C pendant 10 min) . Le signal du plot H est saturant à 250 pixels. On a donc une perte de signal suite à 1 ' incubation de la biopuce fonctionnalisée par le duplex contenant le dommage avec du lysat cellulaire br t. Cette perte de signal est d'environ 30 %, ce qui correspond à la coupure partielle de 1 ' oligonucléotide modifié 0 par les enzymes contenues dans le lysat .The experiment is repeated with lysate denatured (heating at 100 ° C for 10 min). The signal from pad H is saturated at 250 pixels. There is therefore a loss of signal following the incubation of the biochip functionalized by the duplex containing the damage with cell lysate br t. This loss of signal is approximately 30%, which corresponds to the partial cleavage of the oligonucleotide modified 0 by the enzymes contained in the lysate.
Ces expériences sont confirmées par analyse sur gel de polyacrylamide après marquage radioactif de 1 ' oligonucléotide O.These experiments are confirmed by analysis on polyacrylamide gel after radioactive labeling of the oligonucleotide O.
Exemple 3Example 3
Cet exemple illustre la détection de l'activité de réparation d'excision/resynthèse d'un lysat cellulaire total, en utilisant un fragment d'ADN modifié fixé sur un microsupport.This example illustrates the detection of the excision / resynthesis repair activity of a total cell lysate, using a modified DNA fragment fixed on a microcarrier.
Dans ce cas, on utilise le second mode de réalisation du procédé de l'invention. Un fragment d'ADN de 5000 paires de bases est préparé par amplification PCR à partir du phage lambda en utilisant la trousse «Expand™ Long template PCR System» de Roche. Une des amorces d'amplification comporte une séquence de 15 bases (J15) à son extrémité 5' séparée par un synthon aminé de la séquence s'hybridant sur le phage. Cette séquence reste simple brin après l'amplification PCR. Ce fragment PCR est purifié par colonne micro-spin S300 (Amersham- Pharmacia) . Le brin d'ADN est irradié 3 minutes par des rayons ultra-violets de type C { ^ax 254 nm, 0,8 J/cm2).In this case, the second embodiment of the method of the invention is used. A DNA fragment of 5000 base pairs is prepared by PCR amplification from phage lambda using the "Expand ™ Long template PCR System" kit from Roche. One of the amplification primers has a 15 base sequence (J 15 ) at its 5 'end separated by an amino synthon from the sequence hybridizing on the phage. This sequence remains single stranded after PCR amplification. This PCR fragment is purified by an S300 micro-spin column (Amersham-Pharmacia). The DNA strand is irradiated for 3 minutes with ultraviolet rays of type C (^ ax 254 nm, 0.8 J / cm 2 ).
L'ADN irradié est ensuite hybride par l'intermédiaire d'un oligonucléotide de 30 bases de long cIi5cJ15 complémentaire pour une part de la séquence I15 et pour une autre part de la séquence J15, sur une biopuce pb2 (MICAM) comportant quatre séquences oligonucléotidiques différentes sur quatre plots différents (SI, S2 , S3 , I15) , dont la séquence appelée I15. I15 : 5' TTTTT ATC CGT TCT ACA GCCThe irradiated DNA is then hybridized by via an oligonucleotide of 30 bases long cIi 5 cJ 15 complementary for part of the sequence I 15 and for another part of the sequence J 15 , on a pb2 biochip (MICAM) comprising four different oligonucleotide sequences out of four different studs (SI, S2, S3, I 15 ), including the sequence called I 15 . I 15 : 5 'TTTTT ATC CGT TCT ACA GCC
L'hybridation se déroule 1 heure à 37°C dans 20 μl de tampon PBS/NaCl contenant environ 0,1 pmole du produit d'amplification PCR.The hybridization takes place for 1 hour at 37 ° C. in 20 μl of PBS / NaCl buffer containing approximately 0.1 pmole of the PCR amplification product.
La puce est ensuite incubée en présence d'extraits cellulaires totaux dans un tampon adapté comme décrit ci-dessous.The chip is then incubated in the presence of total cell extracts in a suitable buffer as described below.
Expérience d' excision-resynthèse sur la puce fonctionnalisée:Experience of excision-resynthesis on the functionalized chip:
Le protocole est adapté de Robins et al, The EMBO Journal, vol. 10, n°12, pages 3913-3921, 1991 [10] . Une solution est préparée contenant 25 μl de lysat cellulaire de cellules Hela, 10 μl de tampon réactionnel 5X (Hepes KOH 225 mM pH 7,8, KCl, 350 mM, MgCl2 37,5 mM, DTT 4,5 mM, EDTA 2 mM, BSA 0,09 mg/ml, glycérol 17 %) , ATP 2 mM, dGTP 5 μ , dATP 5 μM, dCTP 5 μM, dTTP 1 μM, Biotine-16-2 ' -désoxyuridine-5 ' - triphosphate 4 μM, phosphocréatine 200 mM, créatine phosphokinase type I 12,5 μg dans un volume total de 50 μl . 25 μl de cette solution sont déposés sur la biopuce qui est incubée 2h30 à 30°C en milieu humide. Après rinçage au PBS/NaCl/tween, on effectue la révélation avec la streptavidine- phycoérythrine .The protocol is adapted from Robins et al, The EMBO Journal, vol. 10, n ° 12, pages 3913-3921, 1991 [10]. A solution is prepared containing 25 μl of Hela cell cell lysate, 10 μl of 5X reaction buffer (Hepes KOH 225 mM pH 7.8, KCl, 350 mM, MgCl 2 37.5 mM, DTT 4.5 mM, EDTA 2 mM, BSA 0.09 mg / ml, glycerol 17%), ATP 2 mM, dGTP 5 μ, dATP 5 μM, dCTP 5 μM, dTTP 1 μM, Biotin-16-2 '- deoxyuridine-5' - triphosphate 4 μM , 200 mM phosphocreatine, creatine phosphokinase type I 12.5 μg in a total volume of 50 μl. 25 μl of this solution are deposited on the biochip which is incubated for 2 h 30 min at 30 ° C in a humid environment. After rinsing with PBS / NaCl / tween, we development with streptavidin-phycoerythrin.
La puce est ensuite analysée au microscope.The chip is then analyzed under a microscope.
On observe une forte fluorescence (signal saturant) au niveau du plot fonctionnalisé par 1 ' oligonucléotide I15 Le rapport signal sur bruit (I15/S3) est de 2,2.A strong fluorescence (saturated signal) is observed at the level of the pad functionalized by the oligonucleotide I 15 The signal to noise ratio (I 15 / S3) is 2.2.
Une révélation streptavidine-phycoérythine effectuée avant réparation, par les enzymes contenues dans le lysat, du fragment d'ADN modifié donne un rapport signal sur bruit (I15/S3) de 1.A streptavidin-phycoerythin revelation carried out before repair, by the enzymes contained in the lysate, of the modified DNA fragment gives a signal to noise ratio (I 15 / S3) of 1.
L'expérience est renouvelée avec le fragment non modifié obtenu par réaction PCR. Aucun signal n'est visible sur la puce après réaction d'excision/resynthèse effectuée dans les conditions précédentes (signal/bruit 1) .The experiment is repeated with the unmodified fragment obtained by PCR reaction. No signal is visible on the chip after excision / resynthesis reaction carried out under the preceding conditions (signal / noise 1).
Cet exemple montre que les activités enzymatiques impliquées dans la réparation des bases modifiées de l'ADN sont détectables sur une puce fonctionnalisée par un fragment d'ADN comportant des bases modifiées.This example shows that the enzymatic activities involved in repairing the modified bases of DNA are detectable on a chip functionalized by a DNA fragment comprising modified bases.
Exemple 4Example 4
Cet exemple illustre l'utilisation d'anticorps spécifiques pour la mise en évidence de lésions constituées par des dimères de pyrimidine de type cyclobutaneThis example illustrates the use of specific antibodies for the demonstration of lesions constituted by pyrimidine dimers of cyclobutane type.
On utilise la même biopuce que dans l'exemple 3, sur laquelle est fixée l'ADN irradié dans les mêmes conditions que celles de l'exemple 3, au moyen du même oligonucléotide cl15cJ15. Après lavage en PBS/NaCl/tween, on procède à la révélation des dimères de pyrimidine de type cyclobutane formés par l'irradiation UVB.The same biochip is used as in Example 3, to which the irradiated DNA is fixed under the same conditions as those of Example 3, using the same oligonucleotide cl 15 cJ 15 . After washing with PBS / NaCl / tween, the cyclobutane type pyrimidine dimers formed by UVB irradiation are revealed.
La puce est incubée avec 20 μl de tampon PBS/NaCl contenant 1 μl d'anticorps anti-dimère (500 μg/ml ; Ka iya Biomedicam Company, Seattle, USA) , pendant 1 heure à 37°C.The chip is incubated with 20 μl of PBS / NaCl buffer containing 1 μl of anti-dimer antibody (500 μg / ml; Ka iya Biomedicam Company, Seattle, USA), for 1 hour at 37 ° C.
Après lavage au PBS/NaCl/tween la puce est incubée en présence de 20 μl de PBS/NaCl contenant 1 μl d'anticorps de chèvre anti-souris couplé au marqueurAfter washing with PBS / NaCl / tween the chip is incubated in the presence of 20 μl of PBS / NaCl containing 1 μl of goat anti-mouse antibody coupled to the marker
Cy™3 (1,4 mg/ml; Jackson ImmunoResearch Laboratories,Cy ™ 3 (1.4 mg / ml; Jackson ImmunoResearch Laboratories,
Inc.) pendant 1 h à 37°C.Inc.) for 1 h at 37 ° C.
Après lavage au PBS/NaCl/tween, le signal est enregistré sur la puce. Le rapport signal sur bruit (I15/S3) est de 1,33.After washing with PBS / NaCl / tween, the signal is recorded on the chip. The signal to noise ratio (I 15 / S3) is 1.33.
Ainsi, l'utilisation d'anticorps antidommages, de l'ADN permet de détecter spécifiquement des dommages et donc de suivre leur élimination par des enzymes de réparation.Thus, the use of anti-damage antibodies and DNA makes it possible to specifically detect damage and therefore to monitor its elimination by repair enzymes.
Références citées.References cited.
[1] : Wood et al, Cell, 53, 1988, pages 97-106. [2] : Salles et al, dans Biochimie, 77, 1995, pages 796-802.[1]: Wood et al, Cell, 53, 1988, pages 97-106. [2]: Salles et al, in Biochemistry, 77, 1995, pages 796-802.
[3] : FR-A-2 731 711. [4] : Page et al, Biochemistry, 29, 1990, pages 1016-1024.[3]: FR-A-2 731 711. [4]: Page et al, Biochemistry, 29, 1990, pages 1016-1024.
[5] : Huang et al, Proc. Natl . Acad. Sci., 91, 1994, pages 12213-12217.[5]: Huang et al, Proc. Natl. Acad. Sci., 91, 1994, pages 12213-12217.
[6] : Ro ieu et al, J. Org. Chem. , 63, 1998, pages 5245-5249. [7] : D'Ham et al, Biochemistry, 38, 1999, pages 3335-3344. [8] : Livache et al, Biosens. Bioelectron, 13, 1998, pages 629-634.[6]: Ro ieu et al, J. Org. Chem. , 63, 1998, pages 5245-5249. [7]: D'Ham et al, Biochemistry, 38, 1999, pages 3335-3344. [8]: Livache et al, Biosens. Bioelectron, 13, 1998, pages 629-634.
[9] : ilson III, D.M. and Thompson, L.H. , 1997, Proc.[9]: ilson III, D.M. and Thompson, L.H., 1997, Proc.
Natl. Acad. Sci., 94, pages 12754-12757.Natl. Acad. Sci., 94, pages 12754-12757.
[10] : Sancar, A. (1995) Annu. Rev. Genetics, 29, pages 69-105. [10]: Sancar, A. (1995) Annu. Rev. Genetics, 29, pages 69-105.

Claims

REVENDICATIONS
1. Procédé de détection et de caractérisation d'une ou plusieurs activité (s) de protéine (s) impliquée (s) dans la réparation de l'ADN, comprenant les étapes suivantes : a) fixer sur un support solide au moins un ADN endommagé comportant au moins une lésion connue, b) soumettre cet ADN endommagé à l'action d'une composition de réparation contenant ou non au moins une protéine intervenant pour la réparation de cet ADN endommagé, et c) déterminer l'activité de cette (ces) protéine (s) pour la réparation en mesurant la variation du signal émis par un marqueur qui se fixe sur ou s'élimine du support lors de l'étape b) .1. A method of detecting and characterizing one or more protein activity (ies) involved in DNA repair, comprising the following steps: a) fixing at least one DNA on a solid support damaged comprising at least one known lesion, b) subjecting this damaged DNA to the action of a repair composition containing or not containing at least one protein intervening for the repair of this damaged DNA, and c) determining the activity of this ( ces) protein (s) for repair by measuring the variation of the signal emitted by a marker which is fixed on or removed from the support during step b).
2. Procédé selon la revendication 1, dans lequel la protéine est choisie parmi les protéines impliquées dans la reconnaissance des lésions de l'ADN, les protéines impliquées dans l'excision des lésions de l'ADN, les protéines impliquées dans la resynthèse du (des) nucleotides du brin excisé et les protéines impliquées dans la ligation de brins néoformés .2. Method according to claim 1, in which the protein is chosen from the proteins involved in the recognition of DNA damage, the proteins involved in the excision of DNA damage, the proteins involved in the resynthesis of ( nucleotides of the excised strand and the proteins involved in the ligation of newly formed strands.
3. Procédé selon la revendication 1, dans lequel le marqueur est présent sur l'ADN endommagé fixé sur le support et est éliminé par l'action de la protéine dans l'étape b) .3. Method according to claim 1, in which the marker is present on the damaged DNA fixed on the support and is eliminated by the action of the protein in step b).
4. Procédé selon la revendication 3, dans lequel le marqueur est fixé à une extrémité de l'ADN endommagé, la protéine est une enzyme d'incision ou d'excision des lésions de l'ADN endommagé, et l'incision ou l'excision provoque l'élimination d'un fragment de l'ADN endommagé portant le marqueur.4. Method according to claim 3, in which the marker is attached to one end of the damaged DNA, the protein is an enzyme for incision or excision of lesions of the damaged DNA, and the incision or excision eliminates a fragment of damaged DNA carrying the marker.
5. Procédé selon la revendication 3, dans lequel le marqueur est un marqueur spécifique des lésions de l'ADN endommagé susceptible de révéler ces lésions . avant l'étape b) et la lésion est éliminée lors de la réparation de sorte que le marqueur ne peut plus révéler la lésion.5. Method according to claim 3, in which the marker is a specific marker for lesions of damaged DNA capable of revealing these lesions. before step b) and the lesion is eliminated during the repair so that the marker can no longer reveal the lesion.
6. Procédé selon la revendication 1, dans lequel le marqueur est présent dans la composition de réparation et est introduit dans l'ADN fixé sur le support pendant l'étape b) .6. The method of claim 1, wherein the label is present in the repair composition and is introduced into the DNA attached to the support during step b).
7. Procédé selon la revendication 6, dans lequel la composition de réparation comprend un nucléotide modifié par le marqueur.7. The method of claim 6, wherein the repair composition comprises a nucleotide modified by the label.
8. Procédé selon la revendication 1, dans lequel la composition de réparation est un lysat cellulaire ou une enzyme de réparation purifiée.8. The method of claim 1, wherein the repair composition is a cell lysate or a purified repair enzyme.
9. Procédé selon l'une quelconque des revendications 1 à 8, dans lequel le marqueur est une molécule d'affinité, un composé fluorescent, un anticorps, un haptène ou une biotine.9. Method according to any one of claims 1 to 8, in which the marker is an affinity molecule, a fluorescent compound, an antibody, a hapten or a biotin.
10. Procédé selon l'une quelconque des revendications 1 à 9, dans lequel le support est une biopuce comportant des fragments d'ADN, sur laquelle on fixe par hybridation l'ADN endommagé au moyen d'un oligonucléotide comportant une partie complémentaire de l'ADN endommagé et une partie complémentaire de l'un des fragments d'ADN de la biopuce. 10. Method according to any one of claims 1 to 9, in which the support is a biochip comprising DNA fragments, on which the damaged DNA is fixed by hybridization by means of an oligonucleotide comprising a complementary part of l Damaged DNA and a complementary part of one of the DNA fragments from the biochip.
11. Procédé selon l'une quelconque des revendications 1 à 10, dans lequel le support est une biopuce comportant plusieurs ADN endommagés constitués par des oligonucléotides de séquence identique ou différente, présentant des lésions identiques ou différentes . 11. Method according to any one of claims 1 to 10, in which the support is a biochip comprising several damaged DNAs constituted by oligonucleotides of identical or different sequence, presenting identical or different lesions.
12. Procédé selon l'une quelconque des revendications 1 à 11, dans lequel l'ADN endommagé est un oligonnucleotide court de 15 à 100 bases de long, de préférence de 15 à 50 bases de long, comportant des lésions incorporées dans 1 Oligonucléotide lors de sa synthèse chimique.12. Method according to any one of claims 1 to 11, in which the damaged DNA is a short oligonnucleotide 15 to 100 bases long, preferably 15 to 50 bases long, comprising lesions incorporated into 1 Oligonucleotide during of its chemical synthesis.
13. Procédé selon l'une quelconque des revendications 1 à 11, dans lequel l'ADN endommagé est un polynucléotide de 100 à 20 000 bases de long.13. The method according to any one of claims 1 to 11, wherein the damaged DNA is a polynucleotide 100 to 20,000 bases long.
14. Procédé selon l'une quelconque des revendications 1 à 13, dans lequel l'ADN endommagé est sous forme de simple ou double brin.14. A method according to any one of claims 1 to 13, wherein the damaged DNA is in the form of single or double strand.
15. Biopuce sur laquelle sont fixés plusieurs ADN endommagés constitués par des oligonucléotides de séquence identique ou différente, présentant des lésions identiques ou différentes.15. Biochip on which are fixed several damaged DNAs constituted by oligonucleotides of identical or different sequence, presenting identical or different lesions.
16. Utilisation du procédé selon l'une quelconque des revendications 1 à 14, pour l'étude de la spécificité et la fonctionnalité des protéines impliquées dans la réparation vis-à-vis de lésions connues d'ADN.16. Use of the method according to any one of claims 1 to 14, for the study of the specificity and functionality of proteins involved in repair vis-à-vis known DNA damage.
17. Utilisation du procédé selon l'une quelconque des revendications 1 à 14, pour suivre la cinétique de réparation de lésions de l'ADN par des protéines . 17. Use of the method according to any one of claims 1 to 14, for monitoring the kinetics of repair of DNA damage by proteins.
18. Utilisation du procédé selon l'une quelconque des revendications 1 à 14 , pour évaluer l'effet genotoxique de substances ou d'agents physiques inhibant ou stimulant la synthèse des protéines impliquées dans la réparation de l'ADN.18. Use of the method according to any one of claims 1 to 14, to evaluate the genotoxic effect of substances or physical agents inhibiting or stimulating the synthesis of proteins involved in DNA repair.
19. Utilisation du procédé selon l'une quelconque des revendications 1 à 14, pour le diagnostic de déficiences des protéines de la réparation liées à des maladies. 19. Use of the method according to any one of claims 1 to 14, for the diagnosis of deficiencies in repair proteins linked to diseases.
PCT/FR2001/001605 2000-05-24 2001-05-23 Method for detecting and characterising activity of proteins involved in lesion and dna repair WO2001090408A2 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2001586602A JP4871481B2 (en) 2000-05-24 2001-05-23 Methods for detecting and characterizing the activity of proteins involved in damage and DNA repair
DE60129220T DE60129220T2 (en) 2000-05-24 2001-05-23 DETECTION AND CHARACTERIZATION OF THE ACTIVITY OF PROTEINS INVOLVED IN THE REPAIR OF DNA LESIONS
EP01938324A EP1283911B1 (en) 2000-05-24 2001-05-23 Method for detecting and characterising activity of proteins involved in lesion and dna repair

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR00/06624 2000-05-24
FR0006624A FR2809417B1 (en) 2000-05-24 2000-05-24 DETECTION AND CHARACTERIZATION OF PROTEIN ACTIVITY INVOLVED IN THE REPAIR OF DNA INJURIES

Publications (2)

Publication Number Publication Date
WO2001090408A2 true WO2001090408A2 (en) 2001-11-29
WO2001090408A3 WO2001090408A3 (en) 2002-03-07

Family

ID=8850560

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/FR2001/001605 WO2001090408A2 (en) 2000-05-24 2001-05-23 Method for detecting and characterising activity of proteins involved in lesion and dna repair

Country Status (6)

Country Link
US (1) US20030104446A1 (en)
EP (1) EP1283911B1 (en)
JP (1) JP4871481B2 (en)
DE (1) DE60129220T2 (en)
FR (1) FR2809417B1 (en)
WO (1) WO2001090408A2 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006510385A (en) * 2002-12-20 2006-03-30 コミッサリア　ア　レネルジ　アトミック Method for quantitative evaluation of total DNA repair ability and specific DNA repair ability of at least one biological medium and its application
WO2015049371A1 (en) * 2013-10-03 2015-04-09 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting the responsiveness of a patient affected with chronic myeloid leukemia (cml) to a treatment with a tyrosine kinase inhibitor (tki)
WO2015121596A1 (en) 2014-02-17 2015-08-20 Universite Claude Bernard Lyon 1 Predictive method of characterizing the radiosensitivity and tissular reaction of a patient to therapeutic ionizing radiation
FR3078343A1 (en) * 2018-02-27 2019-08-30 Lxrepair METHOD FOR GENERATING A PROFILE OF REPAIR CAPABILITIES OF TUMOR CELL DNA AND ITS APPLICATIONS
WO2022058472A1 (en) 2020-09-17 2022-03-24 Lxrepair Method for predicting the risk of radiation-induced toxicity
WO2022184907A1 (en) 2021-03-04 2022-09-09 Lxrepair Multiplex quantitative assay for dna double-strand break repair activities in a biological medium and its applications

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140248293A1 (en) 2011-09-08 2014-09-04 Yeda Research And Development Co., Ltd Novel risk biomarkers for lung cancer
EP4014046A1 (en) 2019-08-12 2022-06-22 Yeda Research and Development Co. Ltd Dna repair blood test for predicting response of lung cancer patients to immunotherapy

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2731711A1 (en) * 1995-03-15 1996-09-20 Rech Investissements Sfri Soc METHOD FOR THE QUALITATIVE AND QUANTITATIVE DETECTION OF DNA INJURIES
FR2763600A1 (en) * 1997-05-20 1998-11-27 Genolife METHOD FOR THE QUALITATIVE AND QUANTITATIVE DETECTION OF DNA AND LIGAND ALTERATIONS THEREOF

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5359047A (en) * 1988-09-22 1994-10-25 Massachusetts Institute Of Technology Nucleic acids encoding DNA structure-specific recognition protein and uses therefor
FR2762013B1 (en) * 1997-04-09 1999-06-04 Cis Bio Int DETECTION SYSTEMS FOR NUCLEIC ACID HYBRIDIZATION, PREPARATION METHOD THEREOF AND USES THEREOF
WO1999063828A1 (en) * 1998-06-08 1999-12-16 Emory University Broad specificity dna damage endonuclease
DE19850680A1 (en) * 1998-11-03 2000-05-04 Peter Nehls Method for determining the repair capacity of solutions containing DNA repair enzymes and for detecting DNA structural modifications and base mismatches
AU2397400A (en) * 1998-12-30 2000-07-31 Dana-Farber Cancer Institute, Inc. Mutation scanning array, and methods of use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2731711A1 (en) * 1995-03-15 1996-09-20 Rech Investissements Sfri Soc METHOD FOR THE QUALITATIVE AND QUANTITATIVE DETECTION OF DNA INJURIES
FR2763600A1 (en) * 1997-05-20 1998-11-27 Genolife METHOD FOR THE QUALITATIVE AND QUANTITATIVE DETECTION OF DNA AND LIGAND ALTERATIONS THEREOF

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CALSOU P ET AL: "MEASUREMENTS OF DAMAGE-SPECIFIC DNA INCISION BY NUCLEOTIDE EXCISION REPAIR IN VITRO" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS,US,ACADEMIC PRESS INC. ORLANDO, FL, vol. 202, no. 2, 29 juillet 1994 (1994-07-29), pages 788-795, XP002008620 ISSN: 0006-291X *
D'HAM C ET AL.: "Excision of 5,6-dihydoxy-5,6-dihydrothymine, 5,6-dihydrothymine, and 5-hydroxycytosin from defined sequence oligonucleotides by Escherichia coli endonuclease III and fpg proteins: Kinetic and mechanistic aspects" BOCHEMISTRY, vol. 38, 1999, pages 3335-3344, XP002164194 cité dans la demande *
SALLES B ET AL: "A CHEMILUMINESCENT MICROPLATE ASSAY TO DETECT DNA DAMAGE INDUCED BYGENOTOXIC TREATMENTS" ANALYTICAL BIOCHEMISTRY,US,ACADEMIC PRESS, SAN DIEGO, CA, vol. 232, 1995, pages 37-42, XP002054956 ISSN: 0003-2697 *
SALLES B ET AL: "IN VITRO EUKARYOTIC DNA EXCISION REPAIR ASSAUS: AN OVERVIEW" BIOCHIMIE,FR,PARIS, vol. 77, no. 10, 1995, pages 796-802, XP002054957 ISSN: 0300-9084 cité dans la demande *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006510385A (en) * 2002-12-20 2006-03-30 コミッサリア　ア　レネルジ　アトミック Method for quantitative evaluation of total DNA repair ability and specific DNA repair ability of at least one biological medium and its application
WO2015049371A1 (en) * 2013-10-03 2015-04-09 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting the responsiveness of a patient affected with chronic myeloid leukemia (cml) to a treatment with a tyrosine kinase inhibitor (tki)
WO2015121596A1 (en) 2014-02-17 2015-08-20 Universite Claude Bernard Lyon 1 Predictive method of characterizing the radiosensitivity and tissular reaction of a patient to therapeutic ionizing radiation
WO2015121597A1 (en) 2014-02-17 2015-08-20 Universite Claude Bernard Lyon 1 Predictive method for determining tissual radiosensitivity
FR3078343A1 (en) * 2018-02-27 2019-08-30 Lxrepair METHOD FOR GENERATING A PROFILE OF REPAIR CAPABILITIES OF TUMOR CELL DNA AND ITS APPLICATIONS
WO2019166731A1 (en) 2018-02-27 2019-09-06 Lxrepair Method for generating a profile of the dna repair capabilities of tumour cells and the uses thereof
WO2022058472A1 (en) 2020-09-17 2022-03-24 Lxrepair Method for predicting the risk of radiation-induced toxicity
WO2022184907A1 (en) 2021-03-04 2022-09-09 Lxrepair Multiplex quantitative assay for dna double-strand break repair activities in a biological medium and its applications

Also Published As

Publication number Publication date
EP1283911A2 (en) 2003-02-19
DE60129220T2 (en) 2008-03-13
FR2809417A1 (en) 2001-11-30
EP1283911B1 (en) 2007-07-04
US20030104446A1 (en) 2003-06-05
FR2809417B1 (en) 2004-07-30
JP4871481B2 (en) 2012-02-08
DE60129220D1 (en) 2007-08-16
WO2001090408A3 (en) 2002-03-07
JP2003533994A (en) 2003-11-18

Similar Documents

Publication Publication Date Title
US20060024711A1 (en) Methods for nucleic acid amplification and sequence determination
FR2737502A1 (en) METHOD OF DETECTING NUCLEIC ACIDS USING NUCLEOTIDE PROBES ALLOWING SPECIFIC CAPTURE AND DETECTION
AU2001247328C1 (en) Methods for assay and detection on a microarray
CN113748216B (en) Single-channel sequencing method based on self-luminescence
EP1283911B1 (en) Method for detecting and characterising activity of proteins involved in lesion and dna repair
CN114286867B (en) Method for sequencing polynucleotide based on luminous marker optical signal dynamics and secondary luminous signal
WO1998053099A1 (en) Method for detecting qualitative and quantitative alterations in dna and ligands of said alteration ligands
EP1576191B1 (en) Method for the quantitative assessment of global and specific dna repair capacities of at least one biological medium, and the applications thereof
US20070196832A1 (en) Methods for mutation detection
FR2639358A1 (en) HYBRIDIZATION PROBES AND METHOD FOR DETECTING THE PRESENCE OR ABSENCE OF AT LEAST ONE VARIANT SEQUENCE IN A SINGLE TEST
EP1616031B1 (en) Method of preparing dna fragments by selective fragmentation of nucleic acids and applications thereof
JP2003533994A5 (en)
JPH04504059A (en) Nucleic acid sequencing using chemiluminescent detection
KR102197368B1 (en) Method for detecting polynucleotide using RISC
BE1026457A1 (en) Method and device for the detection and differentiation of point mutations in microorganisms by oligochromatography
WO2003102179A1 (en) Novel method of assyaing nucleic acid using labeled nucleotide
JP2004081062A (en) Nucleic acid probe microarray and assaying method using the same
WO1998045475A1 (en) System for detecting nucleic acid hybridisation, preparation method and application thereof
FR3127504A1 (en) METHOD FOR DETECTION OF RARE MUTATIONS ON LIQUID BIOPSY
BE1011052A3 (en) Diagnostic and/or assay method and kit using sandwich type hybridisation of nucleic acid sequences on solid substrate
FR2920159A1 (en) Identifying, analyzing and/or quantifying alleles of polymorphisms in nucleic acid sample, comprises amplifying double strand of nucleic acid sequence, eliminating heteroduplexes, hybridizing products, and determining and comparing signal
WO2008017761A2 (en) Plastidial microarray
FR2747396A1 (en) METHOD FOR DETECTING A NUCLEIC ACID SEQUENCE FOR ANALYSIS
JP2009240249A (en) Method for detecting nucleic acid sequence and substrate for detecting nucleic acid sequence
FR2866349A1 (en) In vitro diagnosis and prognosis of thrombosis, by amplifying parts of the genes for factors V and II that contain mutations associated with high risk of thrombosis

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR

WWE Wipo information: entry into national phase

Ref document number: 2001938324

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 10258858

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 2001938324

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 2001938324

Country of ref document: EP